Immunogenic composition, use and method of treatment

ABSTRACT

The present invention relates to immunogenic compositions comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, for use in the treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed pursuant to 35 U.S.C. § 371 as a United States National Phase Application of International Application No. PCT/EP2018/058130 filed Mar. 29, 2018 which claims priority from U.S. 62/479,550 filed Mar. 31, 2017.

SEQUENCE LISTING

A sequence listing filed herewith, entitled “PB66320 National Stage seq listing”, prepared Sep. 26, 2019, 274 KB in size, is hereby incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to the field of immunogenic compositions and the use of such compositions in medicine. More particularly, it relates to immunogenic compositions comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, for use in the treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject.

BACKGROUND TO THE INVENTION

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disorder resulting in irreversible decline in lung function as a consequence of inhalation of tobacco smoke or other irritants. Chronic obstructive pulmonary disease (COPD) is recognised as encompassing several conditions (airflow obstruction, chronic bronchitis, bronchiolitis or small airways disease and emphysema) that often coexist (Wilson et al., Eur. Respir. J. 2001; 17: 995-1007). Patients suffer exacerbations of their condition that are usually associated with increased breathlessness, and often have increased cough that may be productive of mucus or purulent sputum (Wilson, Eur Respir J 2001 17:995-1007). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and/or emphysema (Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society. Am J Respir Crit Care Med. 1995 November; 152(5 Pt 2):577-121).

COPD is a major cause of morbidity and mortality worldwide. Approximately one in 20 deaths in 2005 in the US had COPD as the underlying cause. (Drugs and Aging 26:985-999 (2009)). It is projected that in 2020 COPD will rise to the fifth leading cause of disability adjusted life years, chronic invalidating diseases, and to the third most important cause of mortality (Lancet 349:1498-1504 (1997)). The course of COPD is characterized by progressive worsening of airflow limitation and a decline in pulmonary function. COPD may be complicated by frequent and recurrent acute exacerbations (AE), which are associated with enormous health care expenditure and high morbidity. (Proceedings of the American Thoracic Society 4:554-564 (2007)). One study suggests that approximately 50% of acute exacerbations of symptoms in COPD are caused by non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. (Drugs and Aging 26:985-999 (2009)). Haemophilus influenzae (H. influenzae) is found in 20-30% of exacerbations of COPD; Streptococcus pneumoniae, in 10-15% of exacerbations of COPD; and Moraxella catarrhalis, in 10-15% of exacerbations of COPD. (New England Journal of Medicine 359:2355-2365 (2008)). Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis have been shown to be the primary pathogens in acute exacerbations of bronchitis in Hong Kong, South Korea, and the Phillipines, while Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. constitute a large proportion of pathogens in other Asian countries/regions including Indonesia, Thailand, Malaysia and Taiwan (Respirology, (2011) 16, 532-539; doi:10.1111/j.1440.1843.2011.01943.x). In Bangladesh, 20% of patients with COPD showed positive sputum culture for Pseudomonas, Klebsiella, Streptococcus pneumoniae and Haemophilus influenzae, while 65% of patients with AECOPD (acute exacerbation of COPD) showed positive cultures for Pseudomonas, Klebsiella, Acinetobacter, Enterobacter, Moraxella catarrhalis and combinations thereof. (Mymensingh Medical Journal 19:576-585 (2010)). However, it has been suggested that the two most important measures to prevent COPD exacerbation are active immunizations and chronic maintenance of pharmacotherapy. (Proceedings of the American Thoracic Society 4:554-564 (2007)).

One of the difficulties in treating and managing COPD is the heterogeneity of this complex disease in terms of severity, progression, exercise tolerance, and nature of symptoms. This complexity is also evident in acute exacerbations of COPD (AECOPD), which are transient and apparently stochastic periods of increased COPD symptoms requiring additional medical treatment and often hospitalization (Sethi et al., N Eng J Med 2008; 359:2355-65). Known subtypes of exacerbations are defined by the nature of key triggers including bacterial or viral infections, and/or high eosinophil levels, and these events are typically treated with a combination of antibiotics and steroids in a non-specific manner (Bafadhel et al., Am J Respir Crit Care Med 2011; 184:662).

There exists a need for improved uses of immunogenic compositions and methods for the prevention and/or treatment of AECOPD.

SUMMARY OF THE INVENTION

According to the present invention, it has been surprisingly found that bacterial exacerbations are more likely to be repeated in subsequent exacerbations within a subject and that this finding can be used to determine appropriate treatments for a given subject (e.g. a COPD patient).

Accordingly, there is provided in one aspect of the present invention an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, for use in the treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject.

In another aspect of the present invention there is provided an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, for use in a method of treating or preventing a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in a subject, wherein the method comprises identifying that the subject has previously had an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection and then administering the immunogenic composition to the subject.

In another aspect of the present invention there is provided the use of an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, in the manufacture of a medicament for the treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject.

In another aspect of the present invention there is provided the use of an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, in the manufacture of a medicament for use in a method of treating or preventing a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject, wherein the method comprises identifying that the subject has previously had an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection and then administering the immunogenic composition to the subject.

In another aspect of the present invention there is provided a method of treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject at risk of developing an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) recurrence, said method comprising administering to said subject, a therapeutically effective amount of an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof.

In another aspect of the present invention there is provided a method of treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject at risk of developing an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) recurrence, said method comprising administering to said subject, a therapeutically effective amount of an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof, wherein the method comprises identifying that the subject has previously had an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection and then administering the immunogenic composition to the subject.

DETAILED DESCRIPTION

Terminology

A “subject” as used herein is a mammal, including humans, non-human primates, and non-primate mammals such as members of the rodent genus (including but not limited to mice and rats) and members of the order Lagomorpha (including but not limited to rabbits). In one embodiment, the subject is a human.

As used herein, “adjuvant” means a compound or substance that, when administered to a subject in conjunction with a vaccine, immunotherapeutic, or other antigen- or immunogen-containing composition, increases or enhances the subject's immune response to the administered antigen or immunogen (as compared to the immune response that would be obtained in the absence of adjuvant).

As used herein, the term “immunogenic fragment” is a portion of an antigen smaller than the whole, that is capable of eliciting a humoral and/or cellular immune response in a host animal, e.g. human, specific for that fragment. Fragments of a protein can be produced using techniques known in the art, e.g. recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide.

As used herein, the term “conservative amino acid substitution” involves substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in decreased immunogenicity. For example, these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a reference polypeptide.

As used herein “signal peptide” refers to a short (less than 60 amino acids, for example, 3 to 60 amino acids) polypeptide present on precursor proteins (typically at the N terminus), and which is typically absent from the mature protein. The signal peptide (sp) is typically rich in hydrophobic amino acids. The signal peptide directs the transport and/or secretion of the translated protein through the membrane. Signal peptides may also be called targeting signals, transit peptides, localization signals, or signal sequences. For example, the signal sequence may be a co-translational or post-translational signal peptide.

Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by chronic obstruction of lung airflow that interferes with normal breathing and is not fully reversible.

A COPD diagnosis is confirmed by a simple test called spirometry, which measures how deeply a person can breathe and how fast air can move into and out of the lungs. Such a diagnosis should be considered in any patient who has symptoms of cough, sputum production, or dyspnea (difficult or labored breathing), and/or a history of exposure to risk factors for the disease. Where spirometry is unavailable, the diagnosis of COPD should be made using all available tools. Clinical symptoms and signs, such as abnormal shortness of breath and increased forced expiratory time, can be used to help with the diagnosis. A low peak flow is consistent with COPD, but may not be specific to COPD because it can be caused by other lung diseases and by poor performance during testing. Chronic cough and sputum production often precede the development of airflow limitation by many years, although not all individuals with cough and sputum production go on to develop COPD.

An acute exacerbation of COPD (AECOPD) is an acute event characterised by a worsening of the patient's respiratory symptoms that is beyond normal day-to-day variations. Typically an AECOPD leads to a change in medication.

For the purposes of this invention, “treatment of a recurrence of an acute exacerbation of COPD” means ameliorating, stabilising, reducing or eliminating the increased symptoms that are a feature of an acute exacerbation in a subject, wherein the subject has experienced one or more past exacerbations of the same phenotype (e.g. bacterial phenotype associated with a bacterial infection by Haemophilus influenza (e.g. non-typeable H. influenzae (NTHi) and/or Moraxella catarrhalis). As used herein, the phrase “prevention of a recurrence of an acute exacerbation of COPD” means preventing, reducing the incidence or frequency, or reducing the severity of future acute exacerbations of a particular phenotype (e.g. bacterial phenotype associated with a bacterial infection by Haemophilus influenza (e.g. non-typeable H. influenzae (NTHi) and/or Moraxella catarrhalis)) in a subject who has experienced one or more past exacerbations of the same phenotype. In one embodiment, an immunogenic composition according to the present invention is for the reduction of the frequency of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in a subject. In another embodiment, an immunogenic composition according to the present invention is for the reduction of the frequency of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in a subject.

In one embodiment, an immunogenic composition according to the present invention is for the prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in a subject.

In a further embodiment, an immunogenic composition according to the present invention is for the treatment of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in a subject.

DESCRIPTION OF FIGURES

FIG. 1: Flow chart of subject enrollment, sputum sampling, and selection samples for microbiome analysis for AERIS

FIG. 2: Microbiome differences in disease severity and stable or exacerbation visits (A) The Shannon diversity index and relative abundances of bacteria labeled at the phylum and genus level of samples grouped by COPD disease severity. Significant differences in relative abundances between groups are labeled with arrows indicating the relative change in abundance; *P<0.05. (B) The same alpha diversity and relative abundances grouped by stable or exacerbation status. (C) Paired analysis of changes in relative abundances of key genera between matched stable and subsequent exacerbation events; *P<0.05.

FIG. 3: Lung microbiome stability (A) Weighted UniFrac distances measured within and between subjects and comparing stable and exacerbation events; ***P<0.001, ****P<0.0001. (B) Unweighted UniFrac distances measured within and between subjects and comparing stable and exacerbation events; ****P<0.0001. (C) Weighted UniFrac distances for all within subject samples as a function of exacerbation frequency defined by number of exacerbation event and the fraction of samples within an individual taken during an exacerbation. (D) Paired weighted UniFrac distances between exacerbation sample and its previous stable sample from that subject. Exacerbation subtypes labelled as B-Bacterial, V-Viral, E-Eosinophilic, Other, or mixed, *P<0.05.

FIG. 4: Markov chain analysis of transitions between exacerbation states (A) Markov chain analysis from longitudinal exacerbation sampling within individuals identifies non-random transition probabilities for bacterial and eosinophilic exacerbations, but not viral. The size of each node is proportional to abundance of that exacerbation type and the width of the edges are proportional to the transition probabilities. (B) Markov chain analysis of the bacterial exacerbation identifies significantly different transition probabilities for bacterial exacerbations which were positive and negative for the presence of H. influenzae.

FIG. 5: ABCD Assessment Tool for COPD

COPD is characterised by progressive worsening of airflow limitation and a decline in pulmonary function. The disease is complicated by acute exacerbations (AECOPD), which are transient and apparently stochastic periods of increased COPD symptoms requiring additional medical treatment and often hospitalisation. The present inventors have now surprisingly found that an understanding of a subject's exacerbation history (i.e. previous exacerbation phenotype) can be used to guide future therapeutic strategies. Clinical data from a previous exacerbation can be used to inform the likely phenotype of the next acute exacerbation, enabling administration of preventative/prophylactic treatment and/or a more rapid administration of appropriate therapy on presentation of an acute exacerbation.

The present inventors have found, in particular, that bacterial exacerbations are more likely to be repeated in subsequent exacerbations within a subject (e.g. a COPD patient). Thus, COPD patients with a documented history of one or more bacterial exacerbations represent a high-risk sub-population of COPD patients that would benefit from preventive therapy with an immunogenic composition.

As used herein, the term “bacterial exacerbation” refers to an exacerbation associated with a positive bacterial pathogen on routine culture (Haemophilus influenza, Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus aureus or Pseudomonas aeruginosa) or a total aerobic CFU count greater than or equal to 10⁷ cells. In one embodiment, the bacterial exacerbation is associated with a positive bacterial culture for

a) Haemophilus influenza (e.g. non-typeable H. influenzae (NTHi));

b) Moraxella catarrhalis; or

c) Haemophilus influenzae (e.g. non-typeable H. influenzae (NTHi)) and Moraxella catarrhalis

In one aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection is defined by:

a) a positive bacterial pathogen on culture of an induced or spontaneous sputum sample obtained from a subject; and/or

b) a total aerobic CFU count greater than or equal to 10⁷ cells; and/or

c) the presence of increased sputum purulence.

In a further aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection is defined by a positive bacterial pathogen on culture of an induced or spontaneous sputum sample obtained from a subject.

In a further aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection is defined by a total aerobic CFU count greater than or equal to 10⁷ cells.

In a further aspect, the bacterial infection is present in the lung(s) of a subject.

In a further aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the bacterial infection occurred in the presence of Haemophilus influenza (e.g. non-typeable H. influenzae (NTHi)) and/or Moraxella catarrhalis. In one aspect, the bacterial infection occurred in the presence of Haemophilus influenza (e.g. non-typeable H. influenzae (NTHi)). In another aspect, the bacterial infection occurred in the presence of Moraxella catarrhalis.

In one aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is at risk for developing an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection.

In particular, the present inventors found that a significant decrease in entropy of the microbiome (Shannon diviersity index; P_(adj)<0.05) and an increase in the relative abundance of Proteobacteria, such as Haemophilus, is associated with an increase in disease severity. Furthermore, Moraxella showed a significant increase in exacerbation (P=0.0153). Thus, in one aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has a decreased entropy of the lung microbiome as measured according to the Shannon diversity index (P_(adj)<0.05) compared to a measurement taken during a previous acute exacerbation in COPD (AECOPD) in the same subject. In another aspect, there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has a Shannon diversity index less than 3.0.

In another aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has an increased abundance of Proteobacteria, e.g. Haemophilus and/or Moraxella.

The present inventors assessed the contribution of changes in the COPD airway microbiome to the incidence of AECOPD in patients aged 40-85 years with a confirmed diagnosis of COPD, categorised as moderate, severe, or very severe according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification.

The Global Strategy for the Diagnosis, Management and Prevention of COPD prepared by GOLD state that COPD should be considered in any patient with dyspnea, chronic cough or sputum production, and/or a history of exposure to risk factors for the disease, such as tobacco smoking, occupation, or pollutants. A spirometry assessment, measuring airflow limitation, is required to establish diagnosis. The classification of airflow limitation severity in COPD outlined in the GOLD strategy is shown in Table 1.

TABLE 1 Classification of airflow limitation severity in COPD (Based on post-bronchodilator FEV₁) In patients with FEV₁/FVC < 0.70 GOLD 1 Mild FEV₁ ≥ 80% predicted GOLD 2 Moderate 50% ≤ FEV₁ < 80% predicted GOLD 3 Severe 30% ≤ FEV₁ < 50% predicted GOLD 4 Very Severe FEV₁ < 30% predicted

COPD assessment also includes analysis of patient symptoms, and this can be performed using comprehensive disease-specific health status questionnaires such as the Chronic Respiratory Questionnaire (CRQ) and St. George's Respiratory Questionnaire (SGRQ). For routine practice the COPD Assessment Test (CAT™) and The COPD Control Questionnaire (The CCQ©) have been developed. The CAT™ and CCQ© tests do not categorise patients for the purpose of treatment, however for the SRGQ assessment a symptom score ≥25 may be used as the threshold for considered regular treatment for breathlessness. The equivalent threshold for the CAT™ is 10. A simple assessment of breathlessness is the Modified British Medical Research Council (mMRC) Questionnaire.

According to the GOLD strategy, of the patients classified at the GOLD 2 (moderate) stage, approximately 20% may experience frequent exacerbations requiring antibiotic and/or systemic corticosteroid therapy in addition to regular maintenance therapy. The risk of exacerbations is significantly higher for patients classified as GOLD 3 (severe) and GOLD 4 (very severe).

The “ABCD” assessment tool is further used to understand a COPD patient's severity of disease. This assessment combines the patient's spirometry analysis with their exacerbation history and symptom assessment to give a spirometric grade combined with an “ABCD” group. The ABCD assessment tool is shown in FIG. 5.

In another aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has GOLD 2 (moderate), GOLD 3 (severe) or GOLD 4 (very severe) COPD status.

In one aspect there is provided an immunogenic composition, use or method of treatment or prevention according to the present invention, wherein the subject is an adult aged 40-85 years old.

In another aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is a tobacco smoker.

In one aspect, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has experienced at least one (e.g. 2 or more, 3 or more) episodes of acute exacerbation in chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection. In another aspect, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has experienced at least one (e.g. 2 or more, 3 or more) episodes of acute exacerbation in chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection within a period of 12 months.

In a further aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has experienced at least one (e.g. 2 or more, 3 or more) episodes of acute exacerbation in chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in the preceding 12 months.

The present inventors also found that in patients with bronchiectasis, a substantial increase in Haemophilus was observed which was evident in both stable and exacerbation events. Bronchiectasis is a condition in which an area of the bronchial tubes is permanently and abnormally widened (dilated), with accompanying infection. Types include cylindrical, follicular, fusiform, saccular, and varicose, named according to the nature of the dilatations. Examination of the walls of the bronchial tubes reveals destruction of the normal structural elements, with replacement by scar tissue. Pus collects within the bronchi, and the normal flow of oxygen into the lungs, and carbon dioxide out of the lungs (air exchange) is impaired. The bronchi show signs of inflammation, with swelling and invasion by a variety of immune cells. The inflamed areas show signs of increased growth of blood vessels. The area of the lung which should be served by a diseased bronchial tube is also prone to inflammation and infection. The most immediate symptom is persistent coughing with sputum production.

Thus, in another aspect there is provided an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has bronchiectasis.

The present invention also provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject has experienced an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection and failed to achieve resolution of symptoms after antibiotic therapy.

Additional Pharmacologic Therapy

The present invention further provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking one or more other therapeutic agents for COPD. In a further embodiment, the present invention also provides an immunogenic composition, use or method of treatment or prevention according to the present invention, wherein the subject has been prescribed one or more other therapeutic agents for COPD.

Pharmacologic therapy for COPD is utilised to control and reduce symptoms, reduce the frequency and severity of exacerbations and improve tolerance to exercise. Classes of therapeutic agents that can be used to treat COPD include, but are not limited to, beta₂-agonists, anticholinergics, methylxanthines and phosphodiesterase-4 (PDE-4) inhibitors. Anti-inflammatory agents, such as inhaled corticosteroids are also used, typically in combination with a beta₂-agonist and/or an antibcholinergic.

Beta₂agonists include, but are not limited to, short-acting beta₂-agonists such as fenoterol, levalbuterol, salbutamol, terbutaline, and long-acting beta₂-agonists such as arformoterol, formoterol, indacaterol, vilanterol (e.g. vilanterol as the acetate, 1-naphthoate, (R)-mandelate, α-phenylcinnamate or triphenylacetate (trifenatate) salt), olodaterol and salmeterol (e.g. salmeterol xinafoate).

Anticholinergics include, but are not limited to, short-acting anticholinergics such as ipratropium (e.g. ipratropium bromide), oxitropium (e.g. oxitropium bromide), and long-acting anticholinergics such as aclidinium (e.g. aclidinium bromide, glycopyrronium (e.g. glycopyrronium bromide), tiotropium (e.g. tiotropium bromide) and umeclidinium (e.g. umeclidinium bromide).

Methylxanthines include, but are not limited to, aminophylline and theophylline (SR).

It will be clear to the person skilled in the art that, where appropriate, the other therapeutic agents may be used in the form of salts, prodrugs, esters, or solvates (e.g. hydrates) to optimise the activity and/or stability and/or physical characteristics (e.g. solubility) of the therapeutic agent. The additional therapeutic agents may be used in optically pure form and in either amorphous or crystalline form.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking an anticholinergic therapy, for example umeclidinium (e.g. umeclidinium bromide), for COPD.

Combinations of pharmacologic therapies may also be used for the treatment of COPD. In one embodiment, the therapy is a dual combination of a beta₂-agonist and an anticholinergic, such as the combination of vilanterol, or a pharmaceutically acceptable salt or solvate thereof, and umeclidinium, or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the therapy is a combination of vilanterol trifenatate and umeclidinium bromide.

In a further embodiment, the therapy is a dual combination of a beta₂-agonist and an inhaled corticosteroid, such as the combination of vilanterol, or a pharmaceutically acceptable salt or solvate thereof, and fluticasone furoate. In one embodiment, the therapy is a combination of vilanterol trifenatate and fluticasone furoate.

Pharmacologic therapy may also include the combination of three classes of therapeutic agents, such as the combination of a beta₂-agonist, an anticholinergic and an inhaled corticosteroid. In one embodiment, the therapy is a combination of vilanterol, or a pharmaceutically acceptable salt or solvate thereof, umeclidinium, or a pharmaceutically acceptable salt or solvate thereof, and fluticasone furoate. In a further embodiment, the therapy is a combination of vilanterol trifenatate, umeclidinium bromide and fluticasone furoate.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking one or more other therapeutic agents for COPD selected from the group consisting of beta₂-agonists, anticholinergics, methylxanthines, phosphodiesterase-4 (PDE-4) inhibitors and inhaled corticosteroids.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol (e.g. vilanterol trifenatate), and an anticholinergic, for example umeclidinium (e.g. umeclidinium bromide), for COPD.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol (e.g. vilanterol trifenatate), and an inhaled corticosteroid, for example fluticasone furoate, for COPD.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol (e.g. vilanterol trifenatate), an anticholinergic, for example umeclidinium (e.g. umeclidinium bromide), and an inhaled corticosteroid, for example fluticasone furoate, for COPD.

Pharmacologic therapy may be formulated as solutions, suspensions or as dry powder compositions typically for inhalation via a reservoir dry powder inhaler, unit-dose dry powder inhaler, per-metered multi-dose dry powder inhaler, nasal inhaler, pressurised metered dose inhaler, or nebuliser. Representative dry powder inhalers are the DISKHALER™ inhaler device, the DISKUS™ inhalation device, and the ELLIPTA™ inhalation device, marketed by GlaxoSmithKline. The DISKUS™ inhalation device is, for example, described in GB 2242134A, and the ELLIPTA™ inhalation device is, for example, described in WO 2003/061743 A1, WO 2007/012871 A1 and/or WO 2007/068896 A1.

Dry powder compositions may be presented in unit dose form, as capsules, cartridges or commonly blisters. Umeclidinium, for example umeclidinium bromide, may be formulated as a dry powder composition, wherein umeclidinium is to be administered at a dose of 62.5 mcg or 125 mcg once daily, wherein the dose is the amount of the free cation (i.e. umeclidinium).

Vilanterol, for example vilanterol trifenatate, may be formulated as a dry powder composition, wherein vilanterol is to be administered at a dose of 25 mcg once daily, wherein the dose is the amount of the free base (i.e. vilanterol).

Fluticasone furoate may be formulated as a dry powder composition, wherein fluticasone furoate is to be administered at a dose of 50 mcg, 100 mcg, or 200 mcg once daily. In one embodiment, the dose is 100 mcg once daily.

Individual therapeutic agents may be administered sequentially or simultaneously in separate or combined pharmaceutical formulations/compositions. Where appropriate, the individual therapeutic agents may be admixed within the same formulation, and presented as a fixed pharmaceutical combination. In general such formulations/compositions will include pharmaceutical carriers or excipients.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking an anticholinergic therapy, for example umeclidinium bromide at a dose of 62.5 mcg once daily, for COPD. In a further embodiment, the subject is taking the product Incruse™.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol trifenatate at a dose of 25 mcg once daily, and an anticholinergic, for example umeclidinium bromide at a dose of 62.5 mcg once daily, for COPD. In a further embodiment, the subject is taking the product Anoro™.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a vilanterol trifenatate and umeclidinium bromide for COPD, and wherein umeclidinium bromide and vilanterol trifenatate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised umeclidinium bromide (approximately 125 or 62.5 mcg per blister of umeclidinium), lactose monohydrate and magnesium stearate (for example at about 0.6% w/w of the total weight of the dry powder composition), and a second strip contains vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), lactose monohydrate and magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition).

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol trifenatate at a dose of 25 mcg once daily, and an inhaled corticosteroid, for example fluticasone furoate at a dose of 100 mcg once daily, for COPD. In a further embodiment, the subject is taking the product Breo™.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of vilanterol trifenatate and fluticasone furoate for COPD, wherein vilanterol trifenatate and fluticasone furoate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised fluticasone furoate (approximately 100 mcg per blister) and lactose monohydrate, and a second strip contains vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), lactose monohydrate and magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition).

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a beta₂-agonist, for example vilanterol trifenatate at a dose of 25 mcg once daily, an anticholinergic, for example umeclidinium bromide at a dose of 62.5 mcg once daily, and an inhaled corticosteroid, for example fluticasone furoate at a dose of 100 mcg once daily, for COPD.

In one embodiment, the present invention provides an immunogenic composition for use, use of an immunogenic composition or method of treatment or prevention according to the present invention, wherein the subject is taking a combination of a vilanterol trifenatate, umeclidinium bromide and fluticasone furoate for COPD, wherein umeclidinium bromide, vilanterol trifenatate and fluticasone furoate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised umeclidinium bromide (approximately 125 or 62.5 mcg per blister of umeclidinium), vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition) and lactose monohydrate, and a second strip contains a blend of micronized fluticasone furoate (approximately 100 mcg per blister) and lactose monohydrate.

The inhaler device may deliver, when actuated, the contents of a single blister simultaneously from each of the two blister strips. Each blister strip may be a double foil laminate containing 7, 14 or 30 filled blisters per strip.

All COPD patients, in particular those with moderate to severe persistant airflow obstruction classified as either GOLD 2, GOLD 3 or GOLD 4, are prone to bacterial exacerbations and thus may benefit from treatment or prevention with an immunogenic composition of the present invention when added to existing maintenance therapy for COPD. In one embodiment, an immunogenic composition of the present invention may be used in the treatment of human subjects (e.g. patients) with moderate to severe COPD with a previous history of acute exacerbations (AECOPD), for example at least one (e.g. 2 or more, 3 or more) episodes of moderate to severe AECOPD within the last 12 months. In another embodiment, combination of an immunogenic composition of the present invention with existing maintenance therapy may be used in the treatment of human subjects (e.g. patients) with moderate to severe COPD with a previous history of acute exacerbations (AECOPD), for example at least one (e.g. 2 or more, 3 or more) episodes of moderate to severe AECOPD within the last 12 months. Exacerbations are predominantly triggered by viral infections although bacterial infections such as those that occurred in the presence of Haemophilus influenzae and/or Moraxella catarrhalis may also initiate and/or amplify exacerbations.

The addition of an immunogenic composition according to the present invention to a patient's existing maintenance therapy may be complementary, and, for example, may reduce the frequency and/or severity (i.e. mild, moderate or severe) of AECOPD.

Thus, in a further aspect of the invention there is provided a combination therapy comprising:

-   -   (i) one or more therapeutic agents selected from the group         consisting of beta₂-agonists, anticholinergics, methylxanthines,         phosphodiesterase-4 (PDE-4) inhibitors and inhaled         corticosteroids; and     -   (ii) an immunogenic composition comprising an immunogenic         polypeptide from Haemophilus influenzae or an immunogenic         fragment thereof and/or an immunogenic polypeptide from         Moraxella catarrhalis or an immunogenic fragment thereof.

In a further aspect, there is provided a combination therapy for use in the treatment or prevention of COPD (e.g. moderate, severe or very severe COPD) comprising:

-   -   (i) one or more therapeutic agents selected from the group         consisting of beta₂-agonists, anticholinergics, methylxanthines,         phosphodiesterase-4 (PDE-4) inhibitors and inhaled         corticosteroids; and     -   (ii) an immunogenic composition comprising an immunogenic         polypeptide from Haemophilus influenzae or an immunogenic         fragment thereof and/or an immunogenic polypeptide from         Moraxella catarrhalis or an immunogenic fragment thereof.

In one embodiment, the combination therapy or combination therapy for use comprises an anticholinergic as the therapeutic agent.

In a further embodiment, the combination therapy or combination therapy for use comprises two therapeutic agents a beta₂-agonist and an anticholinergic.

In a further embodiment, the combination therapy or combination therapy for use comprises three therapeutic agents: a beta₂-agonist, an anticholinergic and an inhaled corticosteroid.

In a further embodiment, the combination therapy or combination therapy for use comprises two therapeutic agents: a beta₂-agonist and an inhaled corticosteroid.

In a further embodiment, the combination therapy or combination therapy for use comprises umeclidinium (e.g. umeclidinium bromide) as the anticholinergic, optionally at a dose of 62.5 mcg once daily.

In a further embodiment, the combination therapy or combination therapy for use comprises vilanterol (e.g. vilanterol trifenatate) as the beta₂-agonist, optionally at a dose of 25 mcg once daily.

In a further embodiment, the combination therapy or combination therapy for use comprises fluticasone furoate as the inhaled corticosteroid, optionally at a dose of 100 mcg once daily.

In a further embodiment, the combination therapy of combination therapy for use comprises vilanterol trifenatate at a dose of 25 mcg once daily and umeclidinium bromide at a dose of 62.5 mcg once daily.

In a further embodiment, the combination therapy of combination therapy for use comprises vilanterol trifenatate at a dose of 25 mcg once daily, and fluticasone furoate at a dose of 100 mcg once daily.

In a further embodiment, the combination therapy of combination therapy for use comprises vilanterol trifenatate at a dose of 25 mcg once daily, umeclidinium bromide at a dose of 62.5 mcg once daily, and fluticasone furoate at a dose of 100 mcg once daily.

In a further embodiment, in the combination therapy of combination therapy for use umeclidinium bromide, vilanterol trifenatate and/or fluticasone furoate are formulated at dry powder compositions with lactose monohydrate and optionally magnesium stearate and contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler).

In a further embodiment, in the combination therapy of combination therapy for use umeclidinium bromide, vilanterol trifenatate and fluticasone furoate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised umeclidinium bromide (approximately 125 or 62.5 mcg per blister of umeclidinium), vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition) and lactose monohydrate, and a second strip contains a blend of micronized fluticasone furoate (approximately 100 mcg per blister) and lactose monohydrate.

In a further embodiment, in the combination therapy of combination therapy for use umeclidinium bromide and vilanterol trifenatate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised umeclidinium bromide (approximately 125 or 62.5 mcg per blister of umeclidinium), lactose monohydrate and magnesium stearate (for example at about 0.6% w/w of the total weight of the dry powder composition), and a second strip contains vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), lactose monohydrate and magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition).

In a further embodiment, in the combination therapy of combination therapy for use vilanterol trifenatate and fluticasone furoate are contained within the same dry powder inhaler device (e.g. the Ellipta™ Inhaler), wherein the dry powder inhaler device contains two blister strips, wherein one strip contains a blend of micronised fluticasone furoate (approximately 100 mcg per blister) and lactose monohydrate, and a second strip contains vilanterol trifenatate (approximately 25 mcg per blister of vilanterol), lactose monohydrate and magnesium stearate (for example at about 1.0% w/w/based on the total weight of the dry powder composition).

The inhaler device may deliver, when actuated, the contents of a single blister simultaneously from each of the two blister strips. Each blister strip may be a double foil laminate containing 7, 14 or 30 filled blisters per strip.

Typically, the immunogenic composition, which forms part of the combination therapy along with the one or more therapeutic agents, is formulated as herein below described and packaged separately from the one or more therapeutic agents, for sequential or simultaneous administration.

In one embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an immunogenic polypeptide from non-typeable H. influenzae (NTHi) or an immunogenic fragment thereof.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising Protein D or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to Protein D sequence (SEQ ID NO. 1). In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising Protein D or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 2.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising Protein E or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 4. In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an immunogenic fragment of Protein E, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 5.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising PilA or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 6. In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an immunogenic fragment of PilA, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 7.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising Protein E and PilA, wherein Protein E and PilA are present as a fusion protein, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 8. In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising Protein E and PilA, wherein Protein E and PilA are present as a fusion protein, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 9.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an immunogenic polypeptide from M. catarrhalis or an immunogenic fragment thereof.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising UspA2 or an immunogenic fragment thereof.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an immunogenic fragment of UspA2, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a polypeptide selected from the group consisting of MC-001 (SEQ ID NO. 11), MC-002 (SEQ ID NO. 12), MC-003 (SEQ ID NO. 13), MC-004 (SEQ ID NO. 14), MC-005 (SEQ ID NO. 15), MC-006 (SEQ ID NO. 16), MC-007 (SEQ ID NO. 17), MC-008 (SEQ ID NO. 18), MC-009 (SEQ ID NO. 19), MC-010 (SEQ ID NO. 20) or MC-011 (SEQ ID NO. 21) e.g. MC009 SEQ ID NO. 19.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising a pharmaceutically acceptable excipient or carrier.

In a further embodiment, the combination therapy or combination therapy for use comprises an immunogenic composition comprising an adjuvant, e.g. ASO1E.

Immunogenic Proteins and Immunogenic Fragments

Identity between polypeptides may be calculated by various algorithms. For example, the Needle program, from the EMBOSS package (Free software; EMBOSS: The European Molecular Biology Open Software Suite (2000). Trends in Genetics 16(6): 276-277) and the Gap program from the GCG® package (Accelrys Inc.) may be used. This Gap program is an implementation of the Needleman-Wunsch algorithm described in: Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The BLOSUM62 scoring matrix has been used, and the gap open and extension penalties were respectively 8 and 2.

Looking at the computed alignment, identical residues between two compared sequences can be observed. A percentage of identity can be computed by (1) calculating the number of identities divided by the length of the alignment, multiplied by 100 (for example, for the Needle program analysis), (2) calculating the number of identities divided by the length of the longest sequence, multiplied by 100, (3) calculating the number of identities divided by the length of the shortest sequence, multiplied by 100, or (4) calculating the number of identities divided by the number of aligned residues, multiplied by 100 (a residue is aligned if it is in front of another) (for example, for the Gap program analysis).

In one embodiment, sequence identity is calculated over the full length of the reference sequence (e.g. SEQ ID NO. 1 to 21 of the present invention). The immunogenic polypeptides and immunogenic fragments the invention may be derived from an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference sequence (e.g. SEQ ID NO. 1 to 21 of the present invention) which has been modified by the deletion and/or addition and/or substitution of one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids). Amino acid substitution may be conservative or non-conservative. In one aspect, amino acid substitution is conservative. Substitutions, deletions, additions or any combination thereof may be combined in a single variant so long as the variant is an immunogenic polypeptide.

Immunogenic Polypeptides from Non-Typeable H. Influenzae (NTHi) and Immunogenic Fragments

In one aspect of the invention, the immunogenic composition comprises an immunogenic polypeptide from non-typeable H. influenzae (NTHi) or an immunogenic fragment thereof. In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of a polypeptide from non-typeable H. influenzae (NTHi).

In one aspect of the invention, the immunogenic composition comprises Protein D or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to Protein D sequence.

Protein D may be as described in WO91/18926. In an embodiment, the immunogenic composition of the invention comprises protein D from Haemophilus influenzae (PD), for example, protein D sequence from FIG. 9 (FIGS. 9a and 9b together, 364 amino acids) of EP 0594610 (SEQ ID NO: 1). Inclusion of this protein in the immunogenic composition may provide a level of protection against Haemophilus influenzae related otitis media (Pyrmula et al Lancet 367; 740-748 (2006)). Protein D may be used as a full length protein or as a fragment (for example, Protein D may be as described in WO0056360). For example, a protein D sequence may comprise (or consist) of the protein D fragment described in EP0594610 which begins at the sequence SSHSSNMANT (SerSerHisSerSerAsnMetAlaAsnThr) (SEQ ID NO. 3), and lacks the 19 N-terminal amino acids from FIG. 9 of EP0594610, optionally with the tripeptide MDP from NS1 fused to the N-terminal of said protein D fragment (348 amino acids) (SEQ ID NO:2). In one aspect, the protein D or fragment of protein D is unlipidated.

In one aspect of the invention, the immunogenic composition comprises Protein D or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 1. Immunogenic fragments of Protein D comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 1. The immunogenic fragments may elicit antibodies which can bind SEQ ID NO. 1. In another aspect of the invention, the immunogenic composition comprises Protein D or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 2.

In one aspect of the invention, the immunogenic composition comprises Protein E or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to Protein E sequence. In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of Protein E, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to Protein E sequence.

Protein E (PE) is an outer membrane lipoprotein with adhesive properties. It plays a role in the adhesion/invasion of non-typeable Haemophilus influenzae (NTHi) to epithelial cells. (J. Immunology 183: 2593-2601 (2009); The Journal of Infectious Diseases 199:522-531 (2009), Microbes and Infection 10:87-96 (2008)). It is highly conserved in both encapsulated Haemophilus influenzae and non-typeable H. influenzae and has a conserved epithelial binding domain. (The Journal of Infectious Diseases 201:414-419 (2010)). Thirteen different point mutations have been described in different Haemophilus species when compared with Haemophilus influenzae Rd as a reference strain. Its expression is observed on both logarithmic growing and stationary phase bacteria. (WO2007/084053).

Protein E is also involved in human complement resistance through binding vitronectin. (Immunology 183: 2593-2601 (2009)). PE, by the binding domain PKRYARSVRQ YKILNCANYH LTQVR (SEQ ID NO. 1, corresponding to amino acids 84-108 of SEQ ID NO. 4), binds vitronectin which is an important inhibitor of the terminal complement pathway. (J. Immunology 183:2593-2601 (2009)).

As used herein “Protein E”, “protein E”, “Prot E”, and “PE” mean Protein E from H. influenzae. Protein E may consist of or comprise the amino acid sequence of SEQ ID NO. 4 (corresponding to SEQ ID NO. 4 of WO2012/139225A1): (MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA WNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK) as well as sequences with at least or exactly 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity, over the entire length, to SEQ ID NO. 4. In an aspect of the invention, the immunogenic composition comprises Protein E or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 4. Immunogenic fragments of Protein E comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 4. The immunogenic fragments may elicit antibodies which can bind SEQ ID NO. 4.

In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of Protein E, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 5 (corresponding to Seq ID No. 125 of WO2012/139225A1):

SEQ ID NO. 5: Amino acids 20-160 of Protein E I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK

In one aspect of the invention, the immunogenic composition comprises PilA or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to PilA sequence. In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of PilA, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to PilA sequence.

Pilin A (PilA) is likely the major pilin subunit of H. influenzae Type IV Pilus (Tfp) involved in twitching motility (Infection and Immunity, 73: 1635-1643 (2005)). NTHi PilA is a conserved adhesin expressed in vivo. It has been shown to be involved in NTHi adherence, colonization and biofilm formation. (Molecular Microbiology 65: 1288-1299 (2007)).

As used herein “PilA” means Pilin A from H. influenzae. PilA may consist of or comprise the protein sequence of SEQ ID NO. 6 (corresponding to SEQ ID NO. 58 of WO2012/139225A1) (MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ) as well as sequences with 80% to 100% identity to SEQ ID NO. 6. For example, PilA may be at least 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO. 6. In an aspect of the invention, the immunogenic composition comprises PilA or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to Seq ID NO. 6.

For example, immunogenic fragments of PilA comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 6. The immunogenic fragments may elicit antibodies which can bind SEQ ID NO. 6.

In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of PilA, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO. 7 (corresponding to Seq ID No. 127 of WO2012/139225A1):

SEQ ID NO. 7 Amino acids 40-149 of PilA from H. influenzae strain 86-028NP T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ.

Protein E and Pilin A may be presented as a fusion protein (PE-PilA). In another aspect of the invention, the immunogenic composition comprises Protein E and PilA, wherein Protein E and PilA are present as a fusion protein, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to LVL-735 SEQ ID NO. 8 (corresponding to Seq ID No. 194 of WO2012/139225A1).

SEQ ID NO. 8: LVL735 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149): MKYLLPTAAA GLLLLAAQPA MAIQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ

In another aspect of the invention, the immunogenic composition comprises Protein E and PilA, wherein Protein E and PilA are present as a fusion protein, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to LVL-735, wherein the signal peptide has been removed, SEQ ID NO. 9 (Corresponding to Seq ID No. 219 of WO2012/139225A1).

SEQ ID NO. 9: PE-PilA fusion protein without signal peptide IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ

The immunogenicity of Protein E (PE) and Pilin A (PilA) polypeptides may be measured as described in WO2012/139225A1; the contents of which are incorporated herein by reference.

Immunogenic Polypeptides from Moraxella Catarrhalis and Immunogenic Fragments

In one aspect of the invention, the immunogenic composition comprises an immunogenic polypeptide from M. catarrhalis or an immunogenic fragment thereof.

In another aspect of the invention, the immunogenic composition comprises UspA2 or an immunogenic fragment thereof.

In one aspect of the invention, the immunogenic composition comprises UspA2 or an immunogenic fragment thereof, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to UspA2 sequence. In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of UspA2, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to UspA2 sequence.

Ubiquitous surface protein A2 (UspA2) is a trimeric autotransporter that appears as a lollipop-shared structure in electron micrographs (Hoiczyk et al. EMBO J. 19: 5989-5999 (2000)). It is composed of a N-terminal head, followed by a stalk which ends by an amphipathic helix and a C-terminal membrane domain. (Hoiczyk et al. EMBO J. 19: 5989-5999 (2000)). UspA2 contains a very well conserved domain (Aebi et al., Infection & Immunity 65(11) 4367-4377 (1997)), which is recognized by a monoclonal antibody that was shown protective upon passive transfer in a mouse Moraxella catarrhalis challenge model (Helminnen et al. J Infect Dis. 170(4): 867-72 (1994)).

UspA2 has been shown to interact with host structures and extracellular matrix proteins like fibronectin (Tan et al., J Infect Dis. 192(6): 1029-38 (2005)) and laminin (Tan et al., J Infect Dis. 194(4): 493-7 (2006)), suggesting it can play a role at an early stage of Moraxella catarrhalis infection.

UspA2 also seems to be involved in the ability of Moraxella catarrhalis to resist the bactericidal activity of normal human serum. (Attia A S et al. Infect Immun 73(4): 2400-2410 (2005)). It (i) binds the complement inhibitor C4bp, enabling Moraxella catarrhalis to inhibit the classical complement system, (ii) prevents activation of the alternative complement pathway by absorbing C3 from serum and (iii) interferes with the terminal stages of the complement system, the Membrane Attack Complex (MAC), by binding the complement regulator protein vitronectin. (de Vries et al., Microbiol Mol Biol Rev. 73(3): 389-406 (2009)).

As used herein “UspA2” means Ubiquitous surface protein A2 from Moraxella catarrhalis. UspA2 may consist of or comprise the amino acid sequence of SEQ ID NO: 10 from ATCC 25238:

(SEQ ID NO: 1) MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQN ELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIAN LEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKN TQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQN ETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADID NNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKAS SENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIA KNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFA ATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITA LDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRV NPNLAFKAGAAINTSGNKKGSYNIGVNYEF

as well as sequences with at least or exactly 63%, 66%, 70%, 72%, 74%, 75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity, over the entire length, to SEQ ID NO: 10.

UspA2 as described in SEQ ID NO: 10 contains a signal peptide (for example, amino acids 1 to 29 of SEQ ID NO: 10), a laminin binding domain (for example, amino acids 30 to 177 of SEQ ID NO: 10), a fibronectin binding domain (for example, amino acids 165 to 318 of SEQ ID NO: 10) (Tan et al. JID 192: 1029-38 (2005)), a C3 binding domain (for example, amino acids 30 to 539 of SEQ ID NO: 10 (WO2007/018463), or a fragment of amino acids 30 to 539 of SEQ ID NO: 10, for example, amino acids 165 to 318 of SEQ ID NO: 1 (Hallström T et al. J. Immunol. 186: 3120-3129 (2011)), an amphipathic helix (for example, amino acids 519 to 564 of SEQ ID NO: 10 or amino acids 520-559 of SEQ ID NO:10, identified using different prediction methods) and a C terminal anchor domain (for example, amino acids 576 to 630 amino acids of SEQ ID NO: 10 (Brooks et al., Infection & Immunity, 76(11), 5330-5340 (2008)).

In an embodiment, an immunogenic fragment of UspA2 contains a laminin binding domain and a fibronectin binding domain. In an additional embodiment, an immunogenic fragment of UspA2 contains a laminin binding domain, a fibronectin binding domain and a C3 binding domain. In a further embodiment, an immunogenic fragment of UspA2 contains a laminin binding domain, a fibronectin binding domain, a C3 binding domain and an amphipathic helix.

UspA2 amino acid differences have been described for various Moraxella catarrhalis species. See for example, J Bacteriology 181(13):4026-34 (1999), Infection and Immunity 76(11):5330-40 (2008) and PLoS One 7(9):e45452 (2012).

UspA2 may consist of or comprise an amino acid sequence that differs from SEQ ID NO. 10 at any one or more amino acid selected from the group consisting of: AA (amino acid) 30 to 298, AA 299 to 302, AA 303 to 333, AA 334 to 339, AA 349, AA 352 to 354, AA 368 to 403, AA 441, AA 451 to 471, AA 472, AA474 to 483, AA 487, AA 490, AA 493, AA 529, AA 532 or AA 543. UspA2 may consist of or comprise an amino acid sequence that differs from SEQ ID NO: 10 in that it contains an amino acid insertion in comparison to SEQ ID NO. 10. UspA2 may consists of or comprise an amino acid sequence that differs from SEQ ID NO. 10 at any one of the amino acid differences in SEQ ID NO: 22 through SEQ ID NO: 58. For example, SEQ ID NO. 10 may contain K instead of Q at amino acid 70, Q instead of G at amino acid 135 and/or D instead of N at amino acid 216.

TABLE 1 UspA2 amino acid sequences from 38 strains of Moraxalla catarrhalis (SEQ ID NO: 10 and SEQ ID NO: 22-SEQ ID NO: 58). Strain UspA2 sequences ATCC 25238 MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEK (SEQ ID YLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQG NO: 10) LADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEI HAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNI YELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQT EAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDA YAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDR IAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNA ITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGK FNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (630 aa) American MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKY 2933 LLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEH (SEQ ID DRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYT NO: 22) EEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELS GHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQ KADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQT EAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAEL GIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVA IGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (613 aa) American MKTMKLLPLKIAVTSALIIGLGAASTANAQQQLQTETFLPNFLSNDNYDLTDPFYHNMILGDTA 2912 LLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLD (SEQ ID GKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAI NO: 23) KDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNK AQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVE EGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQ NIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSD IKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTK FAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQ AALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGV NYEF (644 aa) American MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDL 2908 QSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPR (SEQ ID KVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQ NO: 24) YLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLESNVE EGLLDLSGRLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQD QYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQA DIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTA IDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITAL DSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTS GNKKGSYNIGVNYEF (591 aa) Finnish MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQQQQQQQQSRTEIFFPNIFFNENHDELDDAYH 307 NIILGDTALLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPV (SEQ ID YQVDYKLDGKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDV NO: 25) TANQQDAIKGLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSI GEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDI KTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALN KASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTE AIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKAD ADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKS ITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAAL GGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (687 aa) Finnish MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQKSPKTETFLPNIFFNEYADDLDTLYHNMILGD 353 TAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLENGV (SEQ ID QKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREV NO: 26) QNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQD QYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALE SNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSE NTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKN QNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGF DGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYG SKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (683 amino acids) Finnish MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGN 358 TALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGGTIIPLDENGKPVYKLDSIVEQG (SEQ ID KTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 27) VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQ DQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKAL ESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI EDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASS ENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGY GSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) Finnish MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQKTKTEVFLPNLFDNDYYDLTDPLYHSMILGD 216 TATLFDQQDNSKSQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTQDT (SEQ ID RTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAI NO: 28) DALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELA QQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADL TKDIKTLENNIEEGLLELSGHLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKAS SENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADA SFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITD LGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGY GSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) Dutch H2 MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGN (SEQ ID TALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKKGDTIIPLDENGKPVYKLDSIVEQG NO: 29) KTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE VQNNIENIYELVQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQ DQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKAL ESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI EDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASS ENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGY GSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (684 amino acids) Dutch F10 MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENL (SEQ ID QDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYKLDGQEP NO: 30) RRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEV QNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLEL SGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLA AYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAK ASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATAD AITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGL FQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (574 amino acids) Norwegian MKTMKLLPLKIAVTSALIVGLGAASTANAQQQPQTETFFPNIFFNENHDALDDVYHNMILGDTA 1 ITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKK (SEQ ID SVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQ NO: 31) NNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLS GRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDA YAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAY NELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI AKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLI EKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVT ALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAV AIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) Norwegian MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGD 13 TAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGV (SEQ ID KKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 32) VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQ DQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTL ENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAY NELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI AKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLI EKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVT ALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAV AIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) Norwegian MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDL 33 QSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPR (SEQ ID KVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQ NO: 33) YLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLENNVE EGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAA YNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKA SAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADA ITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNALDTKVNALDTKVNAFDGRITALDSKV ENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKK GSYNIGVNYEF (587 amino acids) Norwegian MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGD 25 TAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGV (SEQ ID KKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNRE NO: 34) VQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQ DQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTL ENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAY NELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI AKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLI EKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVT ALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAV AIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (678 amino acids) Norwegian MKTMKLLPLKIAVTSALIVGLGAASTANAQVRDKSLEDIEALLGKIDISKLEKEKKQQTELQKY 27 LLLSQYANVLTMEELNKNVEKNTNSIEALGYEIGWLENDIADLEEGVEELTKNQNTLIEKDEEH (SEQ ID DRLIAQNQADIKTLENNVVEELFNLSDRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYT NO: 35) EEVNKTLEKLITNSVKNTDNIDKNKADIQALENNVEEGLLELSGHLIDQKADLTKDIKALESNV EEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENT QNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNK ASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFET LTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTK VDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKS AVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (616 amino acids) Norwegian MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQ 36 DTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVY (SEQ ID SVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNI NO: 36) ENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGHL IDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYA QKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNE LQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIAK NQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEK DKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTAL DTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAI GAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (676 amino acids) BC5SV MKTMKLLPLKIAVTSALIVGLGAASTANAQNGTSTKLKNLKEYAQYLDNYAQYLDDDIDDL (SEQ ID DKEVGELSQNIAKNQANIKDLNKKLSRDIDSLREDVYDNQYEIVNNQADIEKNQDDIKELE NO: 37) NNVGKELLNLSGRLLDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLI DQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLA AYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSE NTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQ DQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAID ANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITA LDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAA INTSGNKKGSYNIGVNYEF (629 amino acids) Norwegian MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQRSPKTETFLPNIFFNEYADDLDTLYHNMI 14 LGDTAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDK (SEQ ID RLDNGVQKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEE NO: 38) SVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQTD IQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNRIKALENNIEEGLLELSGHL IDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQT EAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNEL QDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKAS AANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATA DAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVEN GMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNK KGSYNIGVNYEF (683 amino acids) Norwegian MKTMKLLPLKIAVTSAMIVGLGAASTANAQAQSNRSLDQVQALLRGIDETKIKKEIQQSQQ 3 PELNKYLTFNQLANALNIEELNNNVQKNTQRLDSAATLYGDLSKTVPKSIKENKESIKENK (SEQ ID ESIKENKESIKENKESIKENKESIKENKESITTLTRKSFQNQVDIVRNNASIEDLYAYGQE NO: 39) VAKSIGEIHAYTEEVNKTLENLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQ KADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNV EEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIE DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKA SSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELA QQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKT VIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTK VNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVN PNLAFKAGAAINTSGNKKGSYNIGVNYEF (700 amino acids) Finnish MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYIETTDPLYHGMILGNTA 414 ITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENG (SEQ ID VKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 40) LNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLENN VEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTD IQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALN KASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQK QTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRI AKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNG NAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAA LSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIG VNYEF (676 amino acids) Japanese MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIV Z7476 ENLQDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYK (SEQ ID LDGQEPRRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEE NO: 41) SVQYLNKEVQNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIDQKADIAQNQAN IQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALN KASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQ QTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYN ELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAK VSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAA TADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQ AALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYN IGVNYEF (678 amino acids) Belgian MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKM Z7530 NKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLI (SEQ ID EKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAE NO: 42) RIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTL KNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESN VEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNI EDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSS DIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADA ITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALS GLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVN YEF (613 amino acids) German MKTMKLLPLKIAVTSALIVGLGAASTANAQATNKDITLEDVLKSIEEIDPYELRDYIEYPT Z8063 AIERFLLLSQYGNTLTLEEFDNDIELLDQDVEDLEESVTELAKNQNSLIEQGEAIKEDLQG (SEQ ID LADFVERQEDKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAKSI NO: 43) GEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALESNVEKGLLELSGHLIDQKADID NNINNIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQANIQDLATYNEL QDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI AKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQN TLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKS AVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (589 amino acids) American MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMI O12E LGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLD (SEQ ID SIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANE NO: 44) ESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQT DIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGH LIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQ TEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKA SAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAAT ADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVE NGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGN KKGSYNIGVNYEF (684 amino acids) Greek MKTMKLLPLKIAVTSALIVGLGAASTANAQQQQKTKTEVFLPNLFYNDYIEETDLLYHNMI MC317 LGDTAALVDRQNYSNSQLKFYSNDEESVPDSLLFSKMLNNQQLNGFKAGDIIIPVDANGQV (SEQ ID IYQKDTRVEGGKTRTVLSVTTKIATQQDVDSAYSRGIQGKVNDLDDEMNFLNHDITSLYDV NO: 45) TANQQDDIKGLKKGVKDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLY DFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSG RLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNEL QDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNI AKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQN TLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKS AVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (650 amino acids) American MKTMKLLPLKIAVTSALIVGLGAVSTTNAQAQSRSLDQIQTKLADLAGKIAAGKNGGGQNN V1122 QNNQNDINKYLFLSQYANILTMEELNNNVVKNSSSIETLETDFGWLENDVADLEDGVEELT (SEQ ID KNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSDRLIDQKADIAKNQADIAQNNE NO: 46) SIEELYDFDNEVAEKIGEIHAYTEEVNKTLQDLITNSVKNTDNIDKNKADIDNNINHIYEL AQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQ KADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKA SAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAAT ADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQA ALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNI GVNYEF (616 amino acids) American MKTMKLLPLKIAVTSALIVGLGTASTANAQVASPANQKIQQKIKKVRKELRQDIKSLRNDI P44 DSNTADIGSLNDDVADNQDDILDNQADIAKNQDDIEKNQADIKELDKEVGVLSREIGSLND (SEQ ID DIADNYTDIIDNYTDIIDNQANIAKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDVADN NO: 47) QDDIAKNQADIQTLENNVEEGLLELSGHLLDQKADIDNNINNIYELAQQQDQHSSDIKTLK KNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQEQTEAIDALNKASSENTQ NIAKNSNRIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKA DIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQ TEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIA KNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKVSADTKFAATADAITKNGN AITKNAKSITDLGTKVDAFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPY SVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (668 amino acids) American MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA V1171 IVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ ID RTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 48) EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIK ALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQ DLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKA SSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQT EAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAK NKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNA ITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALS GLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVN YEF (674 amino acids) American MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDIDTLKQDQQKM TTA24 NKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLI (SEQ ID EKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAE NO: 49) RIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTL KNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESN VEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNI EDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSS DIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADA ITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALS GLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVN YEF (613 amino acids) American MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA O35E IVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ ID RTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 50) EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIK ALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIA QNQTDIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQ QDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAI DANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVN AFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPN LAFKAGAAINTSGNKKGSYNIGVNYEF (576 amino acids) American MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMI SP12-6 LGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLD (SEQ ID SIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANE NO: 51) ESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQT DIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGH LIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQ TEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKA SAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAAT ADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVE NGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGN KKGSYNIGVNYEF (684 amino acids) American MKTMKLLPLKIAVTSAMIIGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTA SP12-5 ITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENG (SEQ ID VKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 52) LNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIAQKEDIAQNQTDIQDL ATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQK ADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQ KQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAY NELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLA KASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFA ATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSK VENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTS GNKKGSYNIGVNYEF (686 amino acids) Swedish MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEK BC5 YLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQG (SEQ ID LADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEI NO: 53) HAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNI YELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQT EAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDA YAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDR IAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNA ITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGK FNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (630 amino acids) American MKTMKLLPLKIAVTSALIVGLGAASTANAQAQDRSLEQIQDKLANLVEKIEQAKSQNGQSQ 7169 KDINQYLLLSQYANVLTMEELNNNVVKNSSSIETLDNDIAWLNDDLIDLDKEVGVLSRDIG (SEQ ID SLHDDVAQNQADIKTLKNNVVEELFNLSDRLIDQEADIAQNNESIEDLYDFGREVAESIGE NO: 54) IHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALENDVGKELLNLSGRLIDQKADIDNN INHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDL SGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKA SAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAAT ADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQA ALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNI GVNYEF (616 amino acids) Finnish MKTMKLLPLKIAVTSAMIIGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTA FIN2344 IVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDT (SEQ ID RTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQT NO: 55) EAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNIN HIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSG RLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQ DAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSA ANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATAD AITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAAL SGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGV NYEF (614 amino acids) American MKTMKLPPLKIAVTSAMIIGLGAASTANAQTTETFLPNLFDNDYTETTDPLYHGMILGDTA V1118 ITQDTQYKFYAENGNEVPDSLFFNKILHDQLLNGFKAGDTIIPLDENGKPVYKLDERTENG (SEQ ID VKRKVYSVTTKTATQADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQY NO: 56) LNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDL ATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEECLLELSGHLIDQK ADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANT DRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAIT KNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAA QAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSY NIGVNYEF (679 amino acids) American MKTMKLLPLKIAVTSALIVGLGAASTANAQETLEEVLESIKQINEQDLQDDIGYNSALDRY V1145 LVLSQYGNLLIAKELNENVEKNSNSIAKNSNSIADLEADVGYLAENQNTLIEQNETINQEL (SEQ ID EGITHELESFIAYAHAQDQKNLVNEFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHA NO: 57) YTEEVNKTLENLITNSVKNTDNITKNKADIQALESNVEKELLNLSGRLIDQKADIDNNINH IYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYA QKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAA YNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSEN TQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKAD ADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKN AKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKF NATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF (724 amino acids) American MKTMKLLPLKIAVTSALIVGLGAASTANAQAQARDRSLEDIQALIGNIDVDKIRSQKQKNP V1156 EIFQYLLLNQLSNTLITDELNNNVIKNTNSIETLDNDIAWLNDDLIDLDKEVGVLSRDIGS (SEQ ID LHDDVAQNQADIKTLENNVVEELFNLSDRLIDQEAEIAQNNESIEDLYDFGREVAESIGEI NO: 58) HAHNEAQNETLKDLITNSVKNTDNIDKNKADIQALENNVEEGLLELSGHLIDQKADLTKDI KALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAID ALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANT DRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAIT KNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGL FQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYE F (611 amino acids)

UspA2 may be UspA2 from M. catarrhalis strain ATCC(a US registered trademark) 25238™, American 2933. American 2912, American 2908, Finnish 307, Finnish 353, Finnish 358, Finnish 216, Dutch H2, Dutch F10, Norwegian 1, Norwegian 13, Norwegian 20, Norwegian 25, Norwegian 27, Norwegian 36, BC5SV, Norwegian 14, Norwegian 3, Finish 414, Japanese Z7476, Belgium Z7530, German Z8063, American O12E, Greek MC317, American V1122, American P44, American V1171, American TTA24, American O35E, American SP12-6, American SP12-5, Swedish BC5, American 7169, Finnish FIN2344, American V1118, American V1145 or American V1156. UspA2 may be UspA2 as set forth in any of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 38. UspA2 may be UspA2 from another source which corresponds to the sequence of UspA2 in any one of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58. Corresponding UspA2 sequences may be determined by one skilled in the art using various algorithms. For example, the Gap program or the Needle program may be used to determine UspA2 sequences corresponding to any one of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58.

UspA2 may be a sequence with at least 95% identity, over the entire length, to any of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58. In one embodiment, UspA2 may be a sequence as set forth in an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 or any subset of SEQ ID NO: 1 or SEQ ID NO. 22 through SEQ ID NO:58.

Immunogenic fragments of UspA2 comprise immunogenic fragments of at least 450 contiguous amino acids of SEQ ID NO: 1, 490 contiguous amino acids of SEQ ID NO: 10 (for example, the UspA2 fragment of MC-004 or MC-005), 511 contiguous amino acids of SEQ ID NO: 10 (for example, the UspA2 fragment of construct MC-001, MC-002, MC-003 or MC-004), 534 contiguous amino acids of SEQ ID NO: 10 (for example, the UspA2 fragment of MC-009 or MC-011) or 535 contiguous amino acids of SEQ ID NO: 10 (for example, the UspA2 fragment of MC-007, MC-008 or MC-010). The immunogenic fragments may elicit antibodies which can bind SEQ ID NO: 10.

Immunogenic fragments of UspA2 may comprise immunogenic fragments of at least 450, 490, 511, 534 or 535 contiguous amino acids of SEQ ID NO: 10. Immunogenic fragments of UspA2 may comprise immunogenic fragments of UspA2, for example any of the UspA2 constructs MC-001 (SEQ ID NO. 11), MC-002 (SEQ ID NO. 12), MC-003 (SEQ ID NO. 13), MC-004 (SEQ ID NO. 14), MC-005 (SEQ ID NO. 15), MC-006 (SEQ ID NO. 16), MC-007 (SEQ ID NO. 17), MC-008 (SEQ ID NO. 18), MC-009 (SEQ ID NO. 19), MC-010 (SEQ ID NO. 20) or MC-011 (SEQ ID NO. 21). The immunogenic fragments may elicit antibodies which can bind the full length sequence from which the fragment is derived.

In another aspect of the invention, the immunogenic composition comprises an immunogenic fragment of UspA2, suitably an isolated immunogenic polypeptide with at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a polypeptide selected from the group consisting of MC-001 (SEQ ID NO. 11), MC-002 (SEQ ID NO. 12), MC-003 (SEQ ID NO. 13), MC-004 (SEQ ID NO. 14), MC-005 (SEQ ID NO. 15), MC-006 (SEQ ID NO. 16), MC-007 (SEQ ID NO. 17), MC-008 (SEQ ID NO. 18), MC-009 (SEQ ID NO. 19), MC-010 (SEQ ID NO. 20) or MC-011 (SEQ ID NO. 21) e.g. MC009 SEQ ID NO. 19 (corresponding to Seq ID No. 69 of WO2015/125118A1).

Immunogenicity of UspA2 polypeptides may be measured as described in WO2015/125118A1; the contents of which are incorporated herein by reference.

The immunogenic compositions of the present invention may comprise protein D, PE-PilA and UspA2 for example:

-   -   PD 10 μg/PE-PilA (LVL735 construct, as described in         WO2012/139225) 10 μg/UspA2 (MC009 construct, as described in         WO2015125118) 10 μg/AS01_(E)     -   PD 10 μg/PE-PilA (LVL735 construct, as described in         WO2012/139225) 10 μg/UspA2 (MC009 construct, as described in         WO2015125118) 3.3 μg/AS01_(E)

The above two specific immunogenic compositions were evaluated in a mouse Moraxella catarrhalis lung inflammation model in WO2015125118 (Example 14).

Dosage

The amount of the immunogenic composition which is required to achieve the desired therapeutic or biological effect will depend on a number of factors such as means of administration, the recipient and the type and severity of the condition being treated, and will be ultimately at the discretion of the attendant physician or veterinarian. The present invention provides an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae or an immunogenic fragment thereof and/or an immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof for use in the treatment or prevention of a recurrence of an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject. In one embodiment, one or more previous exacerbations in the same subject were caused wholly or in part by M. catarrhalis. In a further embodiment, one or more previous exacerbations in the same subject were caused wholly or in part by H. influenzae. In general, a typical dose of the immunogenic polypeptide from Moraxella catarrhalis or an immunogenic fragment thereof may be expected to lie in the range of from about 0.001 mg-0.120 mg. More specifically, a typical dose in a human may lie in the range of from about 0.003 mg to about 0.03 mg of protein. In general, a typical dose of the immunogenic polypeptide from H. influenzae or an immunogenic fragment thereof may be expected to lie in the range of from about 0.005 mg to about 0.05 mg. This dose may be administered as a single unit dose. Several separate unit doses may also be administered. For example, separate unit doses may be administered as separate priming doses within the first year of life or as separate booster doses given at regular intervals (for example, every 1, 5 or 10 years).

In a further embodiment, two doses of an immunogenic composition according to the present invention are administered, optionally according to a 0, 2 month vaccination schedule, wherein the second dose is administered about two-months after the first dose (e.g. at the end of the second month or at the beginning or the third month, for example, the first dose on Day 1 and the second dose on Day 61).

Formulations and Adjuvants

Formulations comprising the immunogenic compositions of the invention may be adapted for administration by an appropriate route, for example, by the intramuscular, sublingual, transcutaneous, intradermal or intranasal route. In one embodiment, the immunogenic compositions of the present invention are administered intramuscularly. Such formulations may be prepared by any method known in the art.

The immunogenic compositions of the present invention may additionally comprise an adjuvant. When the term “adjuvant” is used in this specification, it refers to a substance that is administered in conjunction with the immunogenic composition to boost the patient's immune response to the immunogenic component of the composition.

Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes. In one embodiment, the protein may be adsorbed onto aluminium phosphate. In another embodiment, the protein may be adsorbed onto aluminium hydroxide. In a third embodiment, alum may be used as an adjuvant.

Suitable adjuvant systems which promote a predominantly Th1 response include: non-toxic derivatives of lipid A, Monophosphoryl lipid A (MPL) or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with either an aluminum salt (for instance aluminum phosphate or aluminum hydroxide) or an oil-in-water emulsion. In such combinations, antigen and 3D-MPL are contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum-adsorbed antigen (Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1).

AS01 is an Adjuvant System containing MPL (3-O-desacyl-4′-monophosphoryl lipid A), QS21 ((Quillaja saponaria Molina, fraction 21) Antigenics, New York, N.Y., USA) and liposomes. AS01B is an Adjuvant System containing MPL, QS21 and liposomes (50 μg MPL and 50 μg QS21). AS01E is an Adjuvant System containing MPL, QS21 and liposomes (25 μg MPL and 25 μg QS21). In one embodiment, the immunogenic composition or vaccine comprises AS01. In another embodiment, the immunogenic composition or vaccine comprises AS01B or AS01E. In a particular embodiment, the immunogenic composition or vaccine comprises AS01E.

AS02 is an Adjuvant System containing MPL and QS21 in an oil/water emulsion. AS02V is an Adjuvant System containing MPL and QS21 in an oil/water emulsion (50 μg MPL and 50 μg QS21).

AS03 is an Adjuvant System containing α-Tocopherol and squalene in an oil/water (o/w) emulsion. AS03_(A) is an Adjuvant System containing α-Tocopherol and squalene in an o/w emulsion (11.86 mg tocopherol). AS03_(B) is an Adjuvant System containing α-Tocopherol and squalene in an o/w emulsion (5.93 mg tocopherol). AS03_(C) is an Adjuvant System containing α-Tocopherol and squalene in an o/w emulsion (2.97 mg tocopherol). In one embodiment, the immunogenic composition or vaccine comprises AS03.

AS04 is an Adjuvant System containing MPL (50 μg MPL) adsorbed on an aluminum salt (500 μg Al³⁺). In one embodiment, the immunogenic composition or vaccine comprises AS04.

A system involving the use of QS21 and 3D-MPL is disclosed in WO 94/00153. A composition wherein the QS21 is quenched with cholesterol is disclosed in WO 96/33739. An additional adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210. In one embodiment the immunogenic composition additionally comprises a saponin, which may be QS21. The formulation may also comprise an oil in water emulsion and tocopherol (WO 95/17210). Unmethylated CpG containing oligonucleotides (WO 96/02555) and other immunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are also preferential inducers of a TH1 response and are suitable for use in the present invention.

Additional adjuvants are those selected from the group of metal salts, oil in water emulsions, Toll like receptor agonists, (in particular Toll like receptor 2 agonist, Toll like receptor 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof.

Possible excipients include arginine, pluronic acid and/or polysorbate. In a preferred embodiment, polysorbate 80 (for example, TWEEN (a US registered trademark) 80) is used. In a further embodiment, a final concentration of about 0.03% to about 0.06% is used. Specifically, a final concentration of about 0.03%, 0.04%, 0.05% or 0.06% polysorbate 80 (w/v) may be used.

Thus, in one aspect of the invention, the immunogenic composition comprises a pharmaceutically acceptable excipient or carrier.

In another aspect of the invention, the immunogenic composition comprises an adjuvant, e.g. ASO1E.

The present invention provides a method for the treatment or prevention of exacerbations in chronic obstructive pulmonary disease. The exacerbation of COPD may be an acute exacerbation. The method comprises administering to a subject in need thereof a therapeutically effective amount of the immunogenic composition of the invention.

In an additional aspect, the present invention provides a method for the treatment or prevention of a condition or disease caused wholly or in part by Moraxella catarrhalis and/or Haemophilus influenzae.

EXAMPLES Example 1: Lung Microbiome Analysis and Stochastic Modeling of COPD Exacerbations in the AERIS Study

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disorder resulting in irreversible decline in lung function as a consequence of inhalation of tobacco smoke or other irritants (1). One of the difficulties in treating and managing COPD is the heterogeneity of this complex disease in terms of severity, progression, exercise tolerance, and nature of symptoms (2, 3). This complexity is also evident in acute exacerbations of COPD (AECOPD), which are transient and apparently stochastic periods of increased COPD symptoms requiring additional medical treatment and often hospitalization (4). Known subtypes of exacerbations are defined by the nature of key triggers including bacterial or viral infections, and/or high eosinophil levels, and these events are typically treated with a combination of antibiotics and steroids in a non-specific manner (5).

The lung microbiome represents an emerging opportunity to understand COPD heterogeneity and exacerbations. The healthy human lung contains a variety of commensal microbiota throughout the respiratory tract, and these bacteria can show substantial heterogeneity between individuals, across regions within the lung, and over time within an individual (6-8). Alterations in the taxonomic composition of the lung microbiome, known as dysbiosis, have been associated with multiple lung diseases and in particular may play a functional role in disease severity and exacerbations in COPD (6, 9).

Multiple studies have reported differences in the microbiome between healthy and disease states, differences correlated with COPD severity and associated with exacerbation states within an individual, and interactions between the microbiome and host immune response (7, 9-12). Notably, however, many lung microbiome studies have adopted a cross-sectional design which prevents a longitudinal examination of the microbiome to assess the stability of taxa and correlations with clinical traits monitored over long periods of time and covering multiple exacerbations.

The Acute Exacerbation and Respiratory InfectionS in COPD (AERIS) observational cohort study allows for a unique examination of the lung microbiome with a rich set of microbiology and clinical measurements longitudinally observed in stable time points and exacerbation events in 104 patients with COPD (a subset of the full cohort of 127 patients) (13).

Disclosed herein are data from the 16S rRNA sequencing analysis of the AERIS patient cohort. Through integrated analyses using the total AERIS dataset, the present inventors were able to explore the dynamics of the lung microbiome in COPD across multiple clinical visits and to determine the clinical associations of these changes in a deeply phenotyped cohort.

Methods

Study Design

The AERIS study (ClinicalTrials.gov: NCT01360398) was a prospective, observational cohort study based at University Hospital Southampton (UHS). The study protocol has been described in detail (13).

Processing of Sputum Samples

All study procedures for sputum sampling, the detection of exacerbations, and pathogen detection have been described previously (13). Briefly, patients were followed monthly in the stable state and reviewed within 72 hours of onset of AECOPD symptoms. Sputum samples were obtained by spontaneous expectoration or induced and were processed according to standard methods. COPD exacerbation subtypes were determined using previously-defined criteria (5).

16S rRNA Amplification and Sequencing

The V4 hypervariable region of the 16S rRNA gene was amplified with specific primers (515F/806R), including Illumina sequencing adapters and sample-specific barcodes, and sequenced on an Illumina MiSeq desktop sequencer. Sequence data are deposited in NCBI's Sequence Read Archive (PRJNA377739).

16S rRNA Sequence Analysis

Paired-end sequence reads were filtered for quality, assembled using PEAR (14), and then processed using the QIIME pipeline (15).

Statistical Analyses

Comparisons of bacterial taxonomic relative abundances and alpha diversities were performed with a linear mixed model controlling for gender, age, and repeated measures on the same subject within a group. Longitudinal comparisons of relative abundances between stable and exacerbations time points were performed with a paired t-test. Markov chain analysis was performed by counting transitions between adjacent exacerbations with the subtype of exacerbation classifying each state. Statistical analyses were performed using the ‘R’ language and environment (version 3.3.2).

Results

Population and Sampling

Samples for 16S rRNA sequencing were analyzed from 104 subjects with available sputum samples in the first year of the study (FIG. 1). Characteristics of the cohort used for microbiome analysis were similar to those of the full cohort (unpublished observations).

TABLE 2 Characteristics of the cohort for microbiome analysis Characteristic N = 101 Age (years) at enrolment, mean ± SD 67.1 ± 8.4 Female sex, n (%) 42 (41.6%) BMI at enrolment, mean ± SD 27.6 ± 5.4 Current smokers, n (%) 40 (39.6%) Medication for COPD, n (%) 101 (100%) Inhaled corticosteroids, n (%) 94 (93.1%) COPD status, GOLD stage, n (%) Mild 0 (0%) Moderate 45 (44.6%) Severe 40 (39.6%) Very severe 16 (15.8%) Bronchiectasis status, n (%) 10 (9.9%) Number of exacerbations experienced by subject in 12 months, n (%) One exacerbation 31 (22.0%) Two exacerbations 23 (29.1%) Three or more exacerbations 47 (19.7%) FEV₁ after bronchodilator  47.1 ± 12.8 use (% predicted), mean ± SD Table abbreviations: N—number of subjects in the microbiome cohort, n—number of subjects corresponding to characteristic, SD—standard deviation, COPD—chronic obstructive pulmonary disease, GOLD—global initiative for chronic obstructive lung disease, FEV—forced expiratory volume in 1 second.

Lung Microbiome Composition in Stable State

An analysis of the relative abundances of bacterial taxa identified in the set of 584 microbiome samples passing quality control revealed bacteria commonly observed in the lung microbiome with Firmicutes, Proteobacteria, and Bacteroidetes representing the three most abundant phyla and Veillonella, Haemophilus, Streptococcus, Prevotella, and Moraxella representing the five most abundant genera. The number of successfully sequenced microbiome sputum samples averaged 5.7 per subject with 2.1 collected during an exacerbation. The relative abundances of Firmicutes and Bacteroides correlated with higher alpha diversity, while the abundance Proteobacteria had a negative correlation with other taxa and alpha diversity.

We first compared the composition and diversity of the lung microbiome to trends observed in previous studies. As described in other studies describing the lung microbiome in COPD, we observed a shift towards increasing Proteobacteria with increasing disease severity (16,17). More specifically this shift included a significant increase in Haemophilus (Proteobacteria) and decreases in Prevotella (Bacteroidetes) and Veillonella (Firmicutes), as well as decreased Shannon's entropy (P_(adj)<0.05 for each) with increasing disease severity (FIG. 2A).

Changes in the Lung Microbiome in Exacerbation States

When comparing stable and exacerbation samples across all individuals, the differences were less pronounced than differences between disease severities, with no significant changes in alpha diversity measures or main taxa relative abundances with the exception of the genus Moraxella (Proteobacteria) which showed a significant increase in relative abundance in exacerbations (P=0.0134) (FIG. 2B). To confirm these results within longitudinally-sampled individuals we also used a paired t-test to compare matched stable and exacerbation events within an individual. Again, Moraxella showed a significant increase in exacerbation (P=0.0153) (FIG. 2C).

Clinical and microbiology data have been used as biomarkers to stratify subtypes of COPD and AECOPD (5) and some of these have revealed distinct lung microbiome profiles (9). We compared the composition of previously defined exacerbation subtypes characterized by sputum potentially-pathogenic bacterial culture, viral-PCR, or eosinophil percentage. The two most dissimilar exacerbation signatures were bacterial and eosinophilic. Another classification of COPD with a unique microbiome profile is that of bronchiectasis, where we observed a substantial increase in Haemophilus (P=1.2E-5) which was evident in both stable and exacerbation events.

Longitudinal Stability of the Lung Microbiome

Intensive longitudinal sampling within the AERIS study allowed us to assess the relative stability of the lung microbiome within an individual. To analyze temporal microbiome stability, we computed UniFrac distance (weighted and unweighted) between all pairs of microbiome profiles within a subject, and stratified results based on comparisons between stable-stable, stable-exacerbation, and exacerbation-exacerbation comparisons. In all groups, we found UniFrac distance to be significantly lower within an individual compared to distances between individuals (FIG. 3A-B). This result suggests that individuals have somewhat distinct lung microbiomes from each other. Moreover, weighted UniFrac distances were significantly higher for stable-exacerbation and exacerbation-exacerbation comparisons relative to stable-stable comparisons (P<1.0E-3) (comparisons using unweighted UniFrac distance not significant) (FIG. 3B). This measure-specific result suggests that dysbiosis events in the lung may typically result from changes in the relative abundance of pre-existing bacteria (detected by weighted UniFrac) rather than complete removal or appearance of novel species (detected by unweighted UniFrac).

Moreover, given that weighted UniFrac distances involving exacerbations (stable-exacerbation and exacerbation-exacerbation) were higher than stable-stable, it appears exacerbation events are most likely to be associated with dysbiosis within an individual. While the lung microbiome may have a degree of within-subject stability, there remains a large degree of variation between longitudinal samples, especially when comparing an individual's exacerbation events. One possible explanation is that the frequency of exacerbation events experienced by an individual may contribute to destabilization of the lung microbiome, such that frequent exacerbators may be associated with greater dysbiosis than infrequent exacerbators. To evaluate this hypothesis, we analyzed an individual's UniFrac distance as a function of exacerbation frequency. Because of incomplete sampling of all exacerbation events, we conservatively estimated exacerbation frequency in two ways, by counting total reported exacerbation events and by the proportion of microbiome samples obtained from an exacerbation event relative to the total number of microbiome samples obtained for that individual. We found that the lung microbiome became more distinct with greater exacerbation frequency using either definition, affecting bacterial abundance in both stable and exacerbation states (P<1.0E-5, ANOVA) (FIG. 3C). To identify specific taxa associated with exacerbation frequency, we computed the correlation between each taxon's average abundance with exacerbation frequency across subjects. The genus with the highest positive correlation was Moraxella (R=0.23, P=0.016, Pearson), consistent with our observation of its increased abundance in exacerbations relative to stable states. In contrast, the genus Lactobacillus showed the strongest negative correlation with exacerbation frequency (R=−0.37, P=0.02, Pearson).

To test whether any of the AECOPD phenotypes are more likely to experience dysbiosis, we performed a paired analysis comparing the weighted UniFrac distance between each exacerbation type and its previous stable event. Exacerbations with a bacterial component had significantly higher distance compared to viral, eosinophilic, or other exacerbations (P<0.05 for each) (FIG. 3D). These results suggest that dysbiosis is more likely to occur in bacterial exacerbations compared to stable COPD; however, it is not a required characteristic, as many exacerbations show no detectable evidence of dysbiosis.

Stochastic Modeling of COPD Exacerbation Phenotypes

Having observed that some COPD exacerbation phenotypes had different lung microbiome profiles, we utilized the longitudinal attributes of the AERIS study to model the exacerbations experienced by an individual over time as a stochastic process. We employed a Markov chain analysis and defined each exacerbation event as a discrete state of being positive or negative for bacterial, viral, or eosinophilic status independently for each type (inclusive of mixed-type exacerbations). To estimate the state transition probabilities for each Markov model, we counted the number of exacerbations of a given phenotype which were chronologically followed by another exacerbation with the same phenotype (FIG. 4A). We found significantly non-random transition probabilities for the bacterial and eosinophilic Markov models with p-values of 9.25E-11 and 1.42E-3 (chi-square test, df=3), respectively. In contrast, the viral Markov model was not significant (P=0.141). These results indicate that for bacterial and eosinophilic exacerbations, the phenotype of the next exacerbation experienced by an individual may be more likely to repeat the prior exacerbation phenotype than expected by chance. Interestingly, we did not detect a significant difference in the times between exacerbations for any of the Markov transitions.

Next, a Markov model of bacterial exacerbation phenotype was built to examine the potential role of Haemophilus influenzae (Hi). Most of the Haemophilus observed from sputum is likely non-typeable Haemophilus influenzae (NTHi) a non-capsulated bacterium which commonly infects the airways and whose carriage is commonly associated with COPD and inflammation (12). In our analysis of microbiome profiles in different COPD phenotypes, Haemophilus was the dominant genus observed in patients with bronchiectasis, both in stable and exacerbation events. We hypothesized that patients with a positive Hi culture in a positive bacterial exacerbation may be even more likely to repeat an Hi-positive culture in their next exacerbation. The Markov chain of bacterial exacerbations was modified by dividing the bacterial-positive exacerbation state in two separate states of positive and negative Hi. After calculating the new transition probabilities, this Markov chain was non-random (P=1.42E-12, chi-square test, df=5) and the Hi-positive bacterial exacerbations were most likely to repeat a subsequent Hi-positive exacerbation and less likely to transition non-bacterial exacerbation compared to Hi-negative bacterial exacerbations (P=2.62E-4, Fisher's exact test) (FIG. 4B). The repetition of Hi-positive exacerbations suggests a persistence of Haemophilus in certain subtypes of COPD over time and observed through multiple exacerbations. We also expanded the Markov chain analysis of eosinophilic exacerbations by dividing the eosinophilic-positive state into high eosinophilic (>6% in sputum) and moderate eosinophilic (>3% and <6%) positive exacerbations (18). The revised model showed that the higher the eosinophil levels in the exacerbation the more likely it was to repeat the same high eosinophil phenotype (P=0.02, Fisher's exact test).

Discussion

This study has confirmed previous findings of lung microbiome heterogeneity with distinct patterns of bacterial abundance in COPD subtypes (9, 16-17) and for the first time described the stability of the lung microbiome in COPD and the non-random nature of exacerbations experienced by an individual over time. Our analysis shows that grouping samples by specific phenotypes could yield distinct microbiome populations or probabilities of repeating that type of exacerbation. These subtypes underscore the importance of sample size and stratification in generating reproducible results in studying the microbiome of a heterogeneous disease.

Utilizing the unique repeated longitudinal sampling of the AERIS study design, we found that the lung microbiome shows significantly less variation within an individual than between individuals, indicating some degree of temporal stability of an individual's lung microbiome. Nevertheless, we also observed large dysbiosis events within an individual. To characterize these dysbiotic events, we identified individuals with bacterial exacerbations as well as frequent exacerbations as more likely to experience significant changes in lung microbiome patterns. These findings will require further examination to determine the causes and consequences of lung dysbiosis. One appealing model is that lung microbiome composition can functionally drive host inflammatory signals via bacterial proteins or metabolites; specific examples have already been identified and models for their mechanism are being created and tested (9, 11, 19-20).

An appreciation of an individual's lung microbiome may influence the future clinician's choice of appropriate therapy, especially for exacerbations which are commonly treated with antibiotics. The increasing prevalence of recurrent Clostridium difficile infections after antibiotic use has highlighted the risks of disrupting the healthy microbiome when treating primary infections (21). Therefore a move towards selective-spectrum antimicrobials which may be less likely to disturb commensal species and hence minimize treatment-related risk of future infection or exacerbations needs to be explored. The relationship of NTHi with bronchiectasis and its association with repeating bacterial exacerbations may represent a unique treatment challenge. Haemophilus is known to produce biofilms (22) protecting it from the immune system and antibiotics, which may explain its persistence in these COPD subtypes and may offer another avenue for a therapeutic target. Biofilm formation is a component of antibiotic resistance in Moraxella catarrhalis and Pseudomonas aeruginosa, as well (23), indicating potentially common difficulties in eliminating pathogenic bacteria in the lung. The recurring motifs of key pathogenic bacteria such as Moraxella and NTHi identified in this and other COPD lung microbiome studies support the potential of a vaccine or targeted anti-bacterial drug against these pathogens in order to minimize a wider disruption of the lung microbiome.

The ability to model exacerbation phenotypes as stochastic processes has important implications for diagnosis and treatment of AECOPD if the phenotypes of future clinical events can be accurately predicted. Exacerbation events are typically diagnosed and treated as independent phenomena, as and when they are experienced by a patient with COPD (24). If clinical data from the previous exacerbation can inform the likely phenotype of the next event, it can enable a more rapid administration of the appropriate therapy (25). While bacterial and eosinophilic exacerbations are most likely to repeat the same phenotype in our Markov chain model, there is also evidence from other studies that viral infections may predispose the respiratory tract to subsequent secondary bacterial infections (26) indicating additional longitudinal relationships between the infections and colonization of viruses and bacteria.

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SEQUENCES: SEQ ID NO 1: Protein D (364 amino acids) MetLysLeuLysThrLeuAlaLeuSerLeuLeuAlaAlaGlyValLeuAlaGly CysSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIle IleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAla LeuAlaPheAlaGlnGInAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGly ArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPhe ProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIle GlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyr ProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIle GluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIle LysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysVal LeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPhe AsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeu ValGlnLeuIleAlaTyrThrAspTrpLysGluThrGlnGluLysAspProLysGlyTyr TrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLys TyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysPro AspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHis ProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyr AspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGly ValGluPheLeuLysGlyIleLys SEQ ID NO. 2: Protein D fragment with MDP tripeptide from NS1 (348 amino acids) MetAspProSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIle IleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAla LeuAlaPheAlaGlnGlnAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGly ArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPhe ProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIle GlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyr ProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIle GluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIle LysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysVal LeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPhe AsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeu ValGlnLeuIleAlaTyrThrAspTrpLysGluThrGlnGluLysAspProLysGlyTyr TrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLys TyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysPro AspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHis ProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyr AspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGly ValGluPheLeuLysGlyIleLys SEQ ID NO. 3: SerSerHisSerSerAsnMetAlaAsnThr SEQ ID NO. 4: Protein E from H. influenzae MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVH FDA WNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK SEQ ID NO. 5: Amino acids 20-160 of Protein E I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK SEQ ID NO. 6 PilA from H. influenzae MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ SEQ ID NO. 7 Amino acids 40-149 of PilA from H. influenzae strain 86-028NP T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ SEQ ID NO. 8: LVL735 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149) MKYLLPTAAA GLLLLAAQPA MAIQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ SEQ ID NO. 9: PE-PilA fusion protein without signal peptide IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ SEQ ID NO. 11: MC-001 (protein)-(M)(UspA2 amino acids 30-540)(ASHHHHHH) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE AIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANIKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKASHHHHHH SEQ ID NO. 12 MC-002 (Protein)-(M)(UspA2 amino acids 30-540) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE AIDALNKASSENTQNIEDLAAYNELQD AYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKA SADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK SEQ ID NO. 13 MC-003 (Protein)-(M)(UspA2 amino acids 30-540)(H) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE AIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKH SEQ ID NO. 14 MC-004 (Protein)-(M)(UspA2 amino acids 30-540)(HH) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKHH SEQ ID NO. 15 MC-005 (Protein)-(M)(UspA2 amino acids 30-519)(ASHHHHHH) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKDADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSASHHHHHH SEQ ID NO. 16 MC-006 (Protein)-(M)(UspA2 amino acids 30-519) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKS SEQ ID NO. 17 MC-007 (Protein)-(M)(UspA2 amino acids 30-564)(ASHHHHHH) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKVNAFDGRITALDSKVENGMAAQAAASHHHHHH SEQ ID NO. 18 MC-008 (Protein)-(M)(UspA2 30-564)(HH) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKVNAFDGRITALDSWENGMAAQAAHH SEQ ID NO. 19 MC-009 (Protein)-(M)(UspA2 31-564)(HH) MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLED DVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIE DLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADID NNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEA IDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKN QNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDT KVNAFDGRITALDSKVENGMAAQAAHH SEQ ID NO. 20 MC-010 (Protein)-(M)(UspA2 amino acids 30-564) MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLE DDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESI EDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADI DNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTE ATDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALD TKVNAFDGRITALDSWENGMAAQAA SEQ ID NO. 21 MC-011 (Protein)-(M)(UspA2 amino acids 31-540)(ASHHHHHH) MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLED DVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIE DLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADID NNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEA IDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKN QNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDT KASHHHHHH SEQ ID NO: 22 UspA2 American 2933 (613 aa) MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKYLLLNQLAN TLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENN VVEELFNLSGRLIDQEADIAKNNASTEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKAD IDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQK ADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQ NIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKAS AANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTK VDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 23 UspA2 American 2912 (644 aa) MKTMKLLPLKIAVTSALIIGLGAASTANAQQQLQTETFLPNFLSNDNYDLTDPFYHNMILGDTALLDKQDGS QPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLDGKGKKQKRRQVYSVTT KTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAIKDLKKGVKGLNKELKELDKEVGVL SRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALEN NVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQ DAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNIN NIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENK ASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA LSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 24 UspA2 American 2908 (591 aa) MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDLQSNSDQLKF YSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYWDYKLDGKEPRKVYSVTTKIATAEDVATS SYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQYLNKEVQNNIEHIYELAQQQDQHSSD IKTLESNVEKGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADLTKDIKTLESNVEEGLLDLSG RLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIA KAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRIT ALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGS YNIGVNYEF SEQ ID NO: 25 UspA2 Finnish 307 (687 aa) MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQQQQQQQQSRTEIFFPNIFFNENHDELDDAYHNIILGDTA LLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLDGKGKKQKR RQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAIKGLKKGVKGLNKELK ELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNK ADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQD LAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLA AYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDR IAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSIT DLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAV AIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 26 UspA2 Finnish 353 (683 amino acids) MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQKSPKTETFLPNIFFNEYADDLDTLYHNMILGDTAITHDDQ YKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLENGVQKTVYSVTTKTATADDV NSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLK KNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKT LENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYA KQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQ QTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASF ETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGR VTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGY RVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 27 UspA2 Finnish 358 (684 amino acids) MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQEN QYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGGTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADD VNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTL KKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIK TLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAK QQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADAS FETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDG RVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 28 UspA2 Finnish 216 (684 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQKTKTEVFLPNLFDNDYYDLTDPLYHSMILGDTATLFDQQ DNSKSQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTQDTRTKDGKVETVYSVTTKI ATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNE YDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKAD LTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADIAQNQANIQDLAAYNEL QDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQ DAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAWSAANTDRIAKNKA DADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKV DGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGY RVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 29 UspA2 Dutch H2 (684 amino acids) MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQEN QYKFYADDGNGVPDSLLFNKILHDQLLHGFKKGDTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADD VNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIYELVQQQDQHSSDIKTL KKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIK TLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAK QQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADAS FETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDG RVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 30 UspA2 Dutch F10 (574 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENLQDSDDTQL KFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYWDYKLDGQEPRRVYSVTTKIATQDDV DNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIYELAQQQDQHSSDIKTL KKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANN INNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDA NKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQ AALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 31 UspA2 Norwegian 1 (678 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQQQPQTETFFPNIFFNENHDALDDVYHNMILGDTAITQDNQYK FYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRADVEQ SAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKK NVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQ QTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQT EAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEA IDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALD TKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPN LAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 32 UspA2 Norwegian 13 (678 amino acids) MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGDTAITQDN QYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRAD VEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKT LKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHI KTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGR LLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALD TKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPN LAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 33 UspA2 Norwegian 33 (587 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDLQSNSDQLKF YSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPRKVYSVTTKIATAEDVATS SYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQYLNKEVQNNIEHIYELAQQQDQHSSD IKTLESNVEKGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQD QYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKA SADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNALDTKVNALDTKVNAFDGRITAL DSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSY NIGVNYEF SEQ ID NO: 34 UspA2 Norwegian 25 (678 amino acids) MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGDTAITQDN QYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRAD VEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKT LKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHI KTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGR LLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAI DALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTK NQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALD TKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPN LAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 35 UspA2 Norwegian 27 (616 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQVRDKSLEDIEALLGKIDISKLEKEKKQQTELQKYLLLSQYANVL TMEELNKNVEKNTNSIEALGYEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIKTLENNW EELFNLSDRLIDQEADIAKNNASTEELYDFDNEVAERIGEIHAYTEEVNKTLEKLITNSVKNTDNIDKNKADIQ ALENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAY NELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAWSAANTDRIAKN KADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGT KVDGFDSRVTALDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 36 UspA2 Norwegian 36 (676 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQDTQYKFYAE NGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVYSVTTKTATREDVEQSAYS RGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEE GLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADI AKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAID ALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDAL NKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQN TLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKV NALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAF KAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 37 UspA2 BC5SV (629 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQNGTSTKLKNLKEYAQYLDNYAQYLDDDIDDLDKEVGELSQNIA KNQANIKDLNKKLSRDIDSLREDVYDNQYEIVNNQADIEKNQDDIKELENNVGKELLNLSGRLLDQKADIDN NINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAI DALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIQDLAAYNELQDAYAKQQTEAIDA LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALN KASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSD IKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAIT KNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNA TAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 38 UspA2 Norwegian 14 (683 amino acids) MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQRSPKTETFLPNIFFNEYADDLDTLYHNMILGDTAITHDDQ YKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLDNGVQKTVYSVTTKTATADDV NSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLK KNVEEGLLELSGHLIDQKTDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNRIKA LENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYA KQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQ QTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASF ETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGR VTALDTKVNALDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGY RVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 39 UspA2 Norwegian 3 (700 amino acids) MKTMKLLPLKIAVTSAMIVGLGAASTANAQAQSNRSLDQVQALLRGIDETKIKKEIQQSQQPELNKYLTFNQL ANALNIEELNNNVQKNTQRLDSAATLYGDLSKTVPKSIKENKESIKENKESIKENKESIKENKESIKENKESIKE NKESITTLTRKSFQNQVDIVRNNASIEDLYAYGQEVAKSIGEIHAYTEEVNKTLENLITNSVENTNNITKNKAD IQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLT KDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIED LAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLA AYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDR IAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTVIDANKASADTKFAATADAITKNGNAITKNAKSIT DLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGG YGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 40 UspA2 Finnish 414 (676 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYIETTDPLYHGMILGNTAITQDTQYKFYAE NGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRIMSVTTKTATREDVEQSAYS RGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEE GLLELSGHLIDQKADLTKDIKTLENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADI AKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAID ALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDAL NKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQN TLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKV NALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAF KAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 41 UspA2 Japanese Z7476 (678 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENLQDSDDTQL KFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYKLDGQEPRRVYSVTTKIATQDDV DNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIYELAQQQDQHSSDIKTL KKNVEEGLLELSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQ DAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDA YAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADA DASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVN PNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 42 UspA2 Belgian Z7530 (613 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKYLLLNQLAN TLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENN VVEELFNLSGRLIDQEADIAKNNASTEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKAD IDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQK ADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQ NIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKAS AANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTK VDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 43 German Z8063 (589 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQATNKDITLEDVLKSIEEIDPYELRDYIEYPTAIERFLLLSQYGNT LTLEEFDNDIELLDQDVEDLEESVTELAKNQNSLIEQGEAIKEDLQGLADFVERQEDKILQNETSIKKNTQRN LVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAKSIGEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALE SNVEKGLLELSGHLIDQKADIDNNINNIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQAN IQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIA KNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLI TANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSK VENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGV NYEF SEQ ID NO: 44 UspA2 American O12E (684 amino acids) MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQEN QYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADD VNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTL KKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIK TLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAK QQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADAS FETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDG RVTALDTKVNALDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 45 UspA2 Greek MC317 (650 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQQQQKTKTEVFLPNLFYNDYIEETDLLYHNMILGDTAALVDRQN YSNSQLKFYSNDEESVPDSLLFSKMLNNQQLNGFKAGDIIIPVDANGQVIYQKDTRVEGGKTRTVLSVTTKIA TQQDVDSAYSRGIQGKVNDLDDEMNFLNHDITSLYDVTANQQDDIKGLKKGVKDLKKGVKGLNKELKELDK EVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADI QALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAA YNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADI ANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKT AIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENG MAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 46 UspA2 American V1122 (616 amino acids) MKTMKLLPLKIAVTSALIVGLGAVSTTNAQAQSRSLDQIQTKLADLAGKIAAGKNGGGQNNQNNQNDINKYL FLSQYANILTMEELNNNVVKNSSSIETLETDFGWLENDVADLEDGVEELTKNQNTLIEKDEEHDRLIAQNQA DIQTLENNVVEELFNLSDRLIDQKADIAKNQADIAQNNESIEELYDFDNEVAEKIGEIHAYTEEVNKTLQDLIT NSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENN VEEGLLDLSGRLIDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLA AYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDR IAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSIT DLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAV AIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 47 UspA2 American P44 (668 amino acids) MKTMKLLPLKIAVTSALIVGLGTASTANAQVASPANQKIQQKIKKVRKELRQDIKSLRNDIDSNTADIGSLND DVADNQDDILDNQADIAKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDIADNYTDIIDNYTDIIDNQANI AKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDVADNQDDIAKNQADIQTLENNVEEGLLELSGHLLDQKA DIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQEQ TEATDALNKASSENTQNIAKNSNRIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLID QKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDAL NKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQN TLIEKDKEHDKLITANKTAIDANKVSADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDSRVTALDTKV NAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAIN TSGNKKGSYNIGVNYEF SEQ ID NO: 48 UspA2 American V1171 (674 amino acids) MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQ LKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDV EQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALE SNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLID QKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDAL NKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNK ASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTL IEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNA LDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKA GAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 49 UspA2 American T1A24 (613 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDIDTLKQDQQKMNKYLLLNQLAN TLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENN VVEELFNLSGRLIDQEADIAKNNASTEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKAD IDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQK ADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQ NIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKAS AANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTK VDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 50 UspA2 American O35E (576 amino acids) MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQ LKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDV EQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALE SNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLD QKADIAKNQADIAQNQTDIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELA QQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADT KFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQ AALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 51 UspA2 American SP12-6 (684 amino acids) MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQEN QYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLDSIVEQGKTKWYSVTTKTATADD VNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTL KKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIK TLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAY AKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAK QQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADAS FETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDG RVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 52 UspA2 American SP12-5 (686 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQDTQYKFYAE NGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVYSVTTKTATREDVEQSAYS RGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEE GLLELSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIE EGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQY AQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAK QQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADAS FETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDG RVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAG YRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 53 UspA2 Swedish BC5 (630 amino acids) MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNIL ALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSI KKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNK ADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTD IAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASS ENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTL AKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGN AITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAAL GGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 54 UspA2 American 7169 (616 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQAQDRSLEQIQDKLANLVEKIEQAKSQNGQSQKDINQYLLLSQY ANVLTMEELNNNVVKNSSSIETLDNDIAWLNDDLIDLDKEVGVLSRDIGSLHDDVAQNQADIKTLKNNVVEE LFNLSDRLIDQEADIAQNNESIEDLYDFGREVAESIGEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQAL ENDVGKELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIK ALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAY NELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIA KNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDL GTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIG AGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 55 UspA2 Finnish FIN2344 (614 amino acids) MKTMKLLPLKIAVTSAMIIGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQL KFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDVE QSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALES NVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTL ESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNE LQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAWSAANTDRIAKN KADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGT KVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGA GYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 56 UspA2 American V1118 (679 amino acids) MKTMKLPPLKIAVTSAMIIGLGAASTANAQTTETFLPNLFDNDYTETTDPLYHGMILGDTAITQDTQYKFYAE NGNEVPDSLFFNKILHDQLLNGFKAGDTIIPLDENGKPVYKLDERTENGVKRKVYSVTTKTATQADVEQSAYS RGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEK DLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIE ECLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEA IDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAID ALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKN QNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDT KVNALDTKVNAFDGRITALDSWENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNL AFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 57 UspA2 American V1145 (724 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQETLEEVLESIKQINEQDLQDDIGYNSALDRYLVLSQYGNLLIAK ELNENVEKNSNSIAKNSNSIADLEADVGYLAENQNTLIEQNETINQELEGITHELESFIAYAHAQDQKNLVNE FEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAYTEEVNKTLENLITNSVKNTDNITKNKADIQALESNVE KELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDL ATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAA YNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYN ELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIAN NINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAID ANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSWENGMAA QAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF SEQ ID NO: 58 UspA2 American V1156 (611 amino acids) MKTMKLLPLKIAVTSALIVGLGAASTANAQAQARDRSLEDIQALIGNIDVDKIRSQKQKNPEIFQYLLLNQLSN TLITDELNNNVIKNTNSIETLDNDIAWLNDDLIDLDKEVGVLSRDIGSLHDDVAQNQADIKTLENNVVEELFN LSDRLIDQEAEIAQNNESIEDLYDFGREVAESIGEIHAHNEAQNETLKDLITNSVKNTDNIDKNKADIQALEN NVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQ DQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDA YAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAWSAANTDRIAKNKADA DASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDG FDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVN PNLAFKAGAAINTSGNKKGSYNIGVNYEF 

The invention claimed is:
 1. A method of reducing the frequency of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection in a subject at risk of developing an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) recurrence, said method comprising administering to said subject a therapeutically effective amount of an immunogenic composition comprising an immunogenic polypeptide from Haemophilus influenzae capable of eliciting a humoral and/or cellular immune response in a host animal, wherein the immunogenic polypeptide from Haemophilus influenzae is an amino acid sequence at least 96% identical to a reference sequence selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:
 9. 2. The method according to claim 1, wherein the method further comprises identifying that the subject has previously had an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection before administering the immunogenic composition to the subject.
 3. The method according to claim 1, wherein the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection is defined by: a) a positive bacterial pathogen on culture of an induced or spontaneous sputum sample obtained from a subject; and/or b) a total aerobic CFU count greater than or equal to 10⁷ cells; and/or c) the presence of increased sputum purulence.
 4. The method according to claim 1, wherein the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) associated with a bacterial infection is defined by: a) a positive bacterial pathogen on culture of an induced or spontaneous sputum sample obtained from a subject; and/or b) the presence of an increased sputum purulence.
 5. The method according to claim 1, wherein the bacterial infection occurred in the presence of Haemophilus influenza or Moraxella catarrhalis.
 6. The method according to claim 1, wherein the subject has GOLD 2 (moderate), GOLD 3 (severe) or GOLD 4 (very severe) COPD status.
 7. The method according to claim 1, wherein the subject has experienced at least one (e.g. 2 or more, 3 or more) episodes of acute exacerbation in chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection within a period of 12 months.
 8. The method according to claim 1, wherein the subject has experienced at least one (e.g. 2 or more, 3 or more) episodes of acute exacerbation in chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection in the preceding 12 months.
 9. The method according to claim 1, wherein the subject has bronchiectasis.
 10. The method according to claim 1, wherein the subject has experienced an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) resulting from a bacterial infection and failed to achieve resolution of symptoms after antibiotic therapy.
 11. The method according to claim 1, wherein the subject is taking one or more other therapeutic agents for COPD selected from the group consisting of beta2-agonists, anticholinergics, methylxanthines, phosphodiesterase-4 (PDE-4) inhibitors and inhaled corticosteroids.
 12. The method according to claim 2, wherein the subject is a human.
 13. The method according to claim 1 wherein (a) the immunogenic polypeptide from Haemophilus influenzae is selected from (i) Protein D with at least 96% sequence identity to SEQ ID NO: 2, (ii) Protein E with at least 96% sequence identity to SEQ ID NO: 5, (iii) PilA with at least 96% sequence identity to SEQ ID NO: 7, or (iv) Protein E and PilA as a fusion protein with at least 96% sequence identity to SEQ ID NO:
 9. 14. The method according to claim 5 wherein the bacterial infection occurred in the presence of Haemophilus influenza.
 15. The method according to claim 14 wherein the wherein the bacterial infection occurred in the presence of non-typeable Haemophilus influenzae (NTHi)).
 16. The method according to claim 1 wherein the immunogenic composition further comprises an immunogenic polypeptide from Moraxella catarrhalis capable of eliciting a humoral and/or cellular immune response in a host animal, wherein the immunogenic polypeptide from Moraxella catarrhalis is an amino acid sequence at least 96% identical to a reference sequence selected from MC-009 (SEQ ID NO. 19), MC-010 (SEQ ID NO. 20) or MC-011 (SEQ ID NO. 21).
 17. The method according to claim 16 wherein the immunogenic polypeptide from Moraxella catarrhalis is an amino acid sequence at least 96% identical to SEQ ID NO.
 19. 